Protective Effects of Apamin on Acetaminophen-Induced Hepatotoxicity in Mice

Acetaminophen (APAP) overdose can cause severe liver damage, but therapeutic options are limited. Apamin is a natural peptide present in bee venom and has antioxidant and anti-inflammatory properties. Accumulating evidence suggests that apamin has favorable actions in rodent models of inflammatory disorders. Here, we examined the effect of apamin on APAP-evoked hepatotoxicity. Intraperitoneal administration of apamin (0.1 mg/kg) alleviated histological abnormalities and reduced serum levels of liver enzymes in mice injected with APAP. Apamin inhibited oxidative stress through an increase in the amount of glutathione and activation of the antioxidant system. Apamin also attenuated apoptosis with inhibition of caspase-3 activation. Moreover, apamin reduced serum and hepatic levels of cytokines in APAP-injected mice. These effects were accompanied by suppression of NF-κB activation. Furthermore, apamin inhibited chemokine expression and inflammatory cell infiltration. Our results suggest that apamin dampens APAP-evoked hepatotoxicity through inhibiting oxidative stress, apoptosis, and inflammation.


Introduction
Drug-induced liver injury (DILI) can result from taking various drugs, such as chemical agents, biologics, herbs, or other xenobiotics [1,2]. Accumulating evidence suggests that DILI is closely associated with substantial mortality [1]. Acetaminophen (APAP) is a medication commonly used to reduce fever and pain. APAP overdose is a frequent cause of DILI and has become a global health problem [3]. After taking APAP, about 90% is metabolized to non-toxic metabolites, but the remainder is metabolized to N-acetylp-benzoquinone imine (NAPQI) [4]. This metabolite is detoxified by conjugating with glutathione (GSH). APAP overdose leads to GSH depletion and the accumulated NAPQI causes oxidative stress [4]. Oxidative stress induced by NAPQI can cause hepatocyte death and inflammation, leading to liver damage [5]. N-acetylcysteine is the antidote for APAP poisoning and supplements GSH to detoxify NAPQI [6]. However, its use is limited owing to the low efficacy of its delayed use and the narrow therapeutic window [5]. Thus, it is necessary to develop new drugs for APAP-induced hepatotoxicity.
Bee venom has been used to treat numerous medical conditions, including arthritis, musculoskeletal pain, and multiple sclerosis, in traditional medicine [7]. This natural product is composed of various bioactive compounds, including proteins, peptides, and other low-molecular components [7]. Among them, apamin (APM) is one of the main bioactive components of bee venom [8]. This peptide is composed of 18 amino acids and is known to inhibit small-conductance calcium-activated potassium (SK) channels [9]. These channels play a critical role in learning and memory by modulating synaptic plasticity [10].

Histological Analysis, Immunochemistry (IHC), and Immunofluorescence (IF) Staining
Liver tissues were fixed, paraffin-embedded, and sectioned. The slides were stained with hematoxylin and eosin (H&E) stain. The percentage of necrotic area was determined in 10 randomly selected fields (200×) per sample.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Staining
Apoptosis was analyzed using a TUNEL assay kit (Roche Diagnostics, Indianapolis, IN, USA). Positive cells were counted in 10 arbitrarily chosen fields (600×) per sample.

Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted using TRIzol reagent. The PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan) was used to reverse-transcribe the RNA into cDNA. Then, qRT-PCR was conducted using the Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and primers ( Table 1). Relative mRNA levels were normalized to GAPDH mRNA levels.

Statistical Analysis
Data are shown as mean ± SEM. Comparisons between groups were performed using one-way ANOVA analysis with Bonferroni's test. A p value of <0.05 was considered statistically significant.

APM Dampened APAP-Evoked Hepatotoxicity
To determine the effect of APM on APAP-evoked hepatotoxicity, we first performed H&E staining. As expected, APAP-injected mice exhibited increased necrotic area compared to control mice ( Figure 1A,B). However, administration of APM reduced the necrotic area in APAP-injected mice ( Figure 1A,B). Moreover, serum AST and ALT levels, liver injury indicators [27], were increased after APAP treatment ( Figure 1C,D). APM reduced the levels of these indicators in APAP-injected mice ( Figure 1C,D). These results show that APM has an ameliorative effect on APAP-induced hepatotoxicity.
To determine the effect of APM on APAP-evoked hepatotoxicity, we first performed H&E staining. As expected, APAP-injected mice exhibited increased necrotic area compared to control mice ( Figure 1A,B). However, administration of APM reduced the necrotic area in APAP-injected mice ( Figure 1A,B). Moreover, serum AST and ALT levels, liver injury indicators [27], were increased after APAP treatment ( Figure 1C,D). APM reduced the levels of these indicators in APAP-injected mice ( Figure 1C,D). These results show that APM has an ameliorative effect on APAP-induced hepatotoxicity.

APM Suppressed Apoptotic Cell Death
Hepatocyte apoptosis plays a role in APAP-evoked hepatotoxicity [31,32]. The number of cells stained with TUNEL increased after APAP treatment ( Figure 4A,B). However, APM remarkably suppressed the APAP-induced apoptosis ( Figure 4A,B). Cleaved forms of caspase-3 ( Figure 4C,D) and PARP-1 ( Figure 4C,D) were also reduced by APM, indicating that the peptide inhibited caspase-3 pathway.

APM Suppressed Apoptotic Cell Death
Hepatocyte apoptosis plays a role in APAP-evoked hepatotoxicity [31,32]. The number of cells stained with TUNEL increased after APAP treatment ( Figure 4A,B). However, APM remarkably suppressed the APAP-induced apoptosis ( Figure 4A,B). Cleaved forms of caspase-3 ( Figure 4C,D) and PARP-1 ( Figure 4C,D) were also reduced by APM, indicating that the peptide inhibited caspase-3 pathway.

Discussion
In this study, to assess the action of APM on APAP-evoked hepatotoxicity, we first performed histological analysis and measured serum levels of liver enzymes. When hepatocytes are injured, the plasma membrane becomes permeable, allowing the release of intracellular enzymes into the bloodstream. The two most commonly measured liver enzymes associated with clinical liver damage are AST and ALT. Therefore, measurement of serum levels of these enzymes is a good indicator of liver injury [38]. H&E staining showed that APAP-injected mice exhibited increased necrosis compared to con-

Discussion
In this study, to assess the action of APM on APAP-evoked hepatotoxicity, we first performed histological analysis and measured serum levels of liver enzymes. When hepatocytes are injured, the plasma membrane becomes permeable, allowing the release of intracellular enzymes into the bloodstream. The two most commonly measured liver enzymes associated with clinical liver damage are AST and ALT. Therefore, measurement of serum levels of these enzymes is a good indicator of liver injury [38]. H&E staining showed that APAP-injected mice exhibited increased necrosis compared to control mice. Serum levels of the liver enzymes were increased after APAP injection. Importantly, administration of APM significantly reduced necrotic area and serum AST and ALT levels, suggesting that the peptide exerts a protective action on APAP-evoked hepatotoxicity.
APAP overdose causes GSH depletion and NAPQI accumulation. Excessive NAPQI induces oxidative stress, resulting in hepatocyte death and inflammation [5]. A previous study has demonstrated the production of NAPQI in the liver of C57BL/6N mice treated with APAP [39]. APM has been known to possess strong antioxidant property [13,14]. Therefore, we hypothesized that APM can ameliorate APAP-induced hepatotoxicity through inhibiting oxidative stress. In this study, APAP-injected mice exhibited GSH depletion and significant oxidative stress, as reflected by elevated amounts of lipid peroxidation and DNA oxidation products. However, administration of APM significantly restored GSH levels and attenuated oxidative damage in APAP-injected mice. APM significantly reversed the reduction in expression and activity of catalase and SOD2 in APAP-injected mice. These antioxidant enzymes protect the liver from oxidative damage in APAP-induced hepatotoxicity [40][41][42]. Previously, we showed that APM increased renal heme oxygenase-1 (HO-1) levels in mice injected with lipopolysaccharide (LPS) [14]. HO-1 is the enzyme responsible for breaking down heme into components with antioxidant properties [43]. Therefore, the HO-1 upregulation evoked by APM may be involved in its antioxidant and therapeutic effects on LPS-induced kidney injury. Altogether, our findings suggest that APM ameliorates oxidative stress via restoration of hepatic GSH depletion and upregulation of antioxidant enzymes in APAP-injected mice.
Necrosis is generally believed as the major mode of cell death in APAP-induced hepatotoxicity [4,5]. Although there are conflicting results [44], some studies suggest that apoptosis also plays a role in APAP-evoked hepatotoxicity [31,32]. Here, APAP-injected mice exhibited elevated numbers of TUNEL-positive apoptotic cells. However, APM remarkably inhibited hepatocyte apoptosis. Caspase-3 is an important effector caspase that cleaves various cytoplasmic and nuclear proteins, inducing apoptosis. This enzyme is activated by cleavage of the interdomain linker [45]. We found that APM reduced the cleavage of caspase-3 and its substrate PARP-1, indicating that the peptide inhibits caspase-3 activation. Consistent with our findings, APM protected macrophages from apoptosis evoked by oxidized low-density lipoprotein [46]. APM prevents loss of dopaminergic neurons through inhibiting the caspase-dependent mitochondrial apoptotic pathway [47]. Apoptosis can also be triggered by oxidative stress [5]. Therefore, inhibition of apoptosis by APM may be attributed, at least in part, to its antioxidant effect.
Inflammation also contributes to the development and progression of APAP-evoked hepatotoxicity [5]. Administration of APAP induced excessive cytokine generation and extensive inflammatory cell infiltration [33,34]. The pathogenic role of inflammatory responses was also supported by some human data [48][49][50]. In this study, APM decreased serum and hepatic concentrations of cytokines in APAP-injected mice, indicating that APM inhibits systemic and hepatic inflammation in APAP-induced hepatotoxicity. Moreover, APM inhibited NF-κB activation. This signaling cascade plays a pivotal role in APAPevoked inflammatory responses [51,52]. Infiltration of neutrophils and macrophages was also decreased by APM. Consistently, APM reduced mRNA levels of chemokines (CXCL5 and MCP-1) in APAP-injected mice. Similar to our data, many researches have shown the anti-inflammatory effects of APM [13]. APM inhibited NF-κB cascade and cytokine secretion in LPS-treated microglial cells [53]. The peptide also reduced the generation of cytokines with the attenuation of NF-κB activation in inflamed human keratinocytes [54]. Lee et al. showed that APM treatment inhibited proinflammatory cytokine production and inflammasome activation in mice with gouty arthritis [15]. APM also reduced cytokine concentrations and MPO activity in a mouse model of acute pancreatitis [16]. In addition, APM inhibited cytokine production, NF-κB cascade, and inflammatory cell infiltration in endotoxemic mice [14].
In this study, we administered APM to mice 1 h after an APAP overdose. Therefore, this study has limitations in not reflecting a typical clinical situation in which several hours to several days are required for treatment after an APAP overdose. Similar to our study, however, in most previous animal studies, bioactive molecules were administered before or within 3 h of APAP administration [55][56][57]. Indeed, the injury process is known to progress much faster in mice than in humans after an APAP overdose [58]. Nonetheless, further experiments administering APM at longer time intervals after an APAP overdose will be needed to assess its applicability in the clinical setting.
In summary, we showed that APM has a therapeutic action against APAP-evoked hepatotoxicity in mice. APM attenuated oxidative stress via restoration of GSH depletion and upregulation of antioxidant enzymes. Hepatocyte apoptosis and caspase-3 activation was also alleviated by APM. Moreover, APM suppressed cytokine production, NF-κB activation, and inflammatory cell infiltration. We propose that APM might be a potential therapeutic option for APAP toxicity.