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Microbiology and Biotechnology Letters

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Molecular and Cellular Microbiology (MCM)  |  Functional Genomics and Systems Biology

Microbiol. Biotechnol. Lett. 2022; 50(1): 157-163

https://doi.org/10.48022/mbl.2108.08004

Received: August 15, 2021; Revised: December 30, 2021; Accepted: January 4, 2022

The Antimicrobial Insect Peptide CopA3 Blocks Ethanol-Induced Liver Inflammation and Liver Cell Injury in Mice

Ho Kim*

Division of Life Science and Chemistry, College of Natural Science, Daejin University, Pocheon 11159, Republic of Korea

Correspondence to :
Ho Kim,       hokim@daejin.ac.kr

Abstract

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Alcoholic liver disease (ALD), which encompasses alcoholic steatosis, alcoholic hepatitis, and alcoholic cirrhosis, is a major cause of morbidity and mortality worldwide. Although the economic and health impacts of ALD are clear, few advances have been made in its prevention or treatment. We recently demonstrated that the insect-derived antimicrobial peptide CopA3 exerts anti-apoptotic and anti-inflammatory activities in various cell systems, including neuronal cells and colonic epithelial cells. Here, we tested whether CopA3 inhibits ethanol-induced liver injury in mice. Mice were intraperitoneally injected with ethanol only or ethanol plus CopA3 for 24 h and then liver injury and inflammatory responses were measured. Ethanol enhanced the production of proinflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, and IL-10. It also induced hepatocyte apoptosis and ballooning degeneration in hepatocytes. Notably, all these effects were eliminated or significantly reduced by CopA3 treatment. Collectively, our findings demonstrate that CopA3 ameliorates ethanol-induced liver cell damage and inflammation, suggesting the therapeutic potential of CopA3 for treating ethanol-induced liver injury.

Keywords: Antimicrobial peptide (CopA3), insect-derived peptide, ethanol-induced liver injury, inflammation, apoptosis, ballooning degeneration

Graphical Abstract


Introduction

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Alcoholic liver disease (ALD) represents a spectrum of disease states that begin with steatosis, characterized by fat accumulation in hepatocytes [1, 2], and then progress to steatohepatitis accompanied by inflammation [1, 3−5]. Steatosis is known to be caused by the metabolic conversion of alcohol to acetaldehyde and subsequent generation of NADH, which is involved in fatty acid synthesis in hepatocytes [1−3, 6]. In alcoholic hepatitis, the inflammatory cytokines tumor necrosis factor (TNF)-α [7], interleukin (IL)-6 [8], and IL-8 [9] are critical for the initiation and perpetuation of liver injury and cytotoxic hepatomegaly, and act by inducing apoptosis and severe hepatotoxicity [7−9]. Cirrhosis, a late stage of serious liver disease, is marked by fibrosis [1, 2, 10]. These observations, taken together with an extensive body of literature, have established that the liver is the main site of alcohol metabolism and a major target organ of alcohol-induced injury [1−3, 8]. The susceptibility of the liver to alcohol-induced toxicity is attributable to both the high concentrations of alcohol found in the portal blood (versus systemic) [11], as well as the metabolic consequences of ethanol metabolism [12, 13]. ALD is responsible for ~50% of cases of cirrhosis worldwide [14], and is also the second-most common indication for liver transplant in the United States [14, 15]. Despite the economic and health impacts of ALD, few advances have been made in its prevention or treatment.

CopA3 (LLCIALRKK, D-type, disulfide bond-homodimer, 2,110.1 Da), an antimicrobial peptide isolated from the Korean dung beetle [16], has been shown to suppress bacterial toxin-induced apoptosis of colonic epithelial cells and gut inflammation in mice [17]. It also inhibits 6-hydroxy dopamine-induced apoptosis of neural cells [18], and has been reported to inhibit lipopolysaccharide (LPS)-induced activation of macrophages [19], consistent with its observed immune-modulating effects [20]. Collectively, these results support the ability of CopA3 to block apoptosis and inflammation. Based on this concept, we here assessed possible inhibitory effects of CopA3 in an ethanol-induced mouse liver injury model that exhibits inflammation together with apoptosis.

Materials and Methods

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Synthesis of CopA3

The CopA3 peptide ((LLCIAALRKK, D-type, disulfide bond-homodimer, 2,110.1 Da), isolated from the Dung beetle, Copris tripartitus) was synthesized by AnyGen (Korea). The peptides were purified by reverse-phase high-performance liquid chromatography (HPLC) using a Capcell Pak C18 column (Shiseido, Japan) and eluted with a linear gradient of water-acetonitrile (0−80%) containing 0.1% trifluoroacetic acid (45% recovery). To form the interchain disulfide bond of the CopA3, the synthetic peptide was dissolved in acetonitrile-H2O (50/50) solution and then oxidized in an aqueous 0.1 M NK4HCO3 solution (pH 6.0−6.5) for 24 h. The identity of the peptide was confirmed by electrospray ionization (ESI) mass spectrometry (Platform II; Micromass, United Kingdom) [19].

Acute ethanol-induced mouse liver injury and CopA3 treatment

Male C57BL/6J mice, aged 6 weeks, were intraperitoneally (i.p.) injected with CopA3 (3 mg/kg) and then 1 h later were treated with ethanol (3 g/kg; i.p.) for 24 h [21−23]. Mice in the control group were i.p.-injected with an equal volume of 0.9% saline. Liver tissue samples were either stored in TRIzol reagent (Life Technologies, USA) or 4% formalin solution (w/v) in phosphate-buffered saline (PBS) for further analysis. This study was approved by the Animal Care and Use Committee of Daejin University (ACUC, Korea).

Interleukin-6 (IL-6) and tumor necrosis Factor-α (TNF-α) measurement

To evaluate liver inflammation levels, liver tissues were washed in cold PBS and homogenized in cold PBS, centrifuged (11,000 ×g, 10 min at 4℃), and supernatants were collected. The supernatants were used for measuring mouse IL-6 and TNF-α by ELISA using R&D Systems (USA) [9].

Quantitative RT-PCR analysis of mRNAs

Major proinflammatory cytokines related to alcohol-induced liver inflammation [16, 24] were monitored. Briefly, total RNA was isolated from mouse liver tissue samples using the TRIzol reagent, and quality control tests were performed as instructed by the provider. Expression of mRNAs was stringently determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) based on the following criteria: fold change > ±2, p < 0.05, and a cycle threshold (Ct) value < 35. The following targets were amplified by PCR using the indicated primer pairs: TNF-α, 5'-CGGCAGAGAGGAGGTTGACTTTCT-3' (sense) and 5'-CACAGAAAGCATGATCCGCGACGT-3' (antisense); mouse IL-1β, 5'-CTTCTTTGGGTATTGCTTGGGATC-3' (sense) and 5'-CCAGCTTCAAATCTCACAGCAG-3' (antisense); mouse IFN-γ, 5'-TGCAGGATTTTCATGTCACCAT-3' (sense) and 5'-TGGCATAGATGTGGAAGAAAAGAG-3' (antisense); mouse IL-10, 5'-CAGCAGACTCAATACACACT-3' (sense) and 5'-TGGCCCAGAAATCAAGGAGC-3' (antisense). Mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as a control using the primers 5'-TCTCCATGGTGGTGAAGACA-3' (sense) and 5'-ACTCCACTCACGGCAAATTC-3' (antisense).

Histological analysis

For hematoxylin and eosin (H&E) (Sigma Aldrich, USA) staining, mouse liver tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, and cut into sections. For analysis of mouse liver apoptosis, terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assays were performed on mouse liver sections (5 μm thickness) using a commercial kit (Sigma Aldrich) according to the manufacturer’s suggested protocol. The histopathologists conducting histological analyses were blinded to group-identifying information [25].

Statistical analysis

The results are presented as mean values ± SEM. Data were analyzed using the SIGMA-STAT professional statistics software package (Jandel Scientific Software, USA). Analyses of variance with protected t-tests were used for intergroup comparisons.

Results and Discussion

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CopA3 protects against ethanol-induced inflammation in mice

The pathogenesis of ethanol-related liver disease is known to mainly involve inflammation [21, 26, 27]. In alcoholic hepatitis, the inflammatory cytokines, TNF-α and IL-8, are known to induce liver injury [8, 9]. Elevated serum levels of TNF-α and IL-8 have also been reported in patients with alcoholic hepatitis [21]. Serum TNF-α is increased in patients with ALD and is correlated with mortality [28]. After chronic alcohol consumption, Kupffer cells exhibit enhanced sensitivity to LPS-stimulated TNF-α production [29]. On the basis of these observations, we tested whether CopA3 exerts antiinflammatory activity against acute ethanol-induced mouse liver injury [21, 26, 27]. To this end, we first administered CopA3 (3 mg/kg, i.p.) and then 1 h later injected mice with ethanol (3 g kg, i.p.) to induce liver injury (Fig. 1A). After allowing injury to develop for 24 h, we examined liver sections for inflammation using enzyme-linked immunoassays (ELISAs). As shown in Fig. 1, ethanol exposure significantly increased production of the proinflammatory cytokines, TNF-α, and IL-8, in mouse liver. Notably, pretreatment with CopA3 completely abrogated alcohol-induced upregulation of these cytokines. CopA3 treatment alone did not affect production levels of TNF α or IL-8 (Figs. 1B and C).

Figure 1.CopA3 attenuates ethanol-induced inflammation in mouse livers. (A) Mice in the experimental group (n = 6) were pretreated with CopA3 (30 mg/kg, i.p.) and after 1 h were treated with ethanol (EtOH; 3.0 g/kg, i.p.) for 24 h. Controls included mice injected with 0.9% saline only (saline), saline + EtOH (EtOH) or saline + CopA3 (CopA3). (B, C) Liver tissues were washed and homogenized in cold PBS and centrifuged at 11,000 ×g for 10 min at 4℃. Supernatants were collected and assayed for TNF-α (B) and IL-8 in (C) by ELISA. Results are presented as means ± SEM (n = 6 mice/group; p < 0.05, ethanol-treated group vs. ethanol+CopA3 treatment group).

CopA3 reduces mRNA expression of proinflammatory cytokines in mice injected with ethanol

Like TNF-α, IL-10 is a cytokine that plays a role in reducing alcoholic liver injury and inflammation [8, 9]. IL-10 is released together with TNF-α and other cytokines by Kupffer cells after alcohol consumption [30]. Among inflammatory cytokines, interferon (IFN)-γ is known to induce liver injury in a rat model of alcoholic liver disease [30, 31]. Thus, we next investigated the involvement of these cytokines. As expected, ethanol exposure significantly increased expression of mRNAs for TNF-α (Fig. 2A), IL-1β (Fig. 2B), IFN-γ (Fig. 2C), and IL-10 (Fig. 2D) in mouse liver. Strikingly, these effects were completely blocked by pretreatment with CopA3 (Figs. 2A−D), which again had no effect on hepatic inflammation when administered alone (Figs 2A−D). We speculate that this reduction in ethanol-induced proinflammatory cytokine production by CopA3 is attributable to the anti-inflammatory activity of CopA3 [19, 20]. Indeed, it has been reported that CopA3 inhibits LPS-induced activation of macrophages, markedly decreasing proinflammatory cytokine production, nitric oxide synthesis, and phagocytosis [19, 20].

Figure 2.CopA3 reduces ethanol-induced proinflammatory cytokine expression in mouse livers. (A-D) Mice in the experimental group (n = 6) were pretreated with CopA3 (30 mg/kg, i.p.) and after 1 h were treated with ethanol (EtOH; 3.0 g/kg, i.p.) for 24 h. Controls included mice injected with 0.9% saline only (saline), saline + EtOH (EtOH) or saline + CopA3 (CopA3). mRNA levels of TNF-α (A), IL-1β (B), IFN-γ (C) and IL-10 (D) in liver tissues were determined by RT-PCR. Results are presented as means ± SEM (n = 6 mice/group; *, p < 0.05, ethanol-treated group vs. ethanol+CopA3 treatment group).

CopA3 blocks ethanol-induced hepatocyte apoptosis in mice exposed to ethanol

Given that the pathogenesis of ethanol-related liver disease mainly involves hepatocyte apoptosis, an important determinant of subsequent inflammation and fibrosis [21, 26, 27], we measured the inhibitory effect of CopA3 on ethanol-induced hepatocyte apoptosis by performing TUNEL assays, which detect DNA fragmentation (an apoptotic cell marker) [32]. Paraffin-embedded liver sections were also stained with H&E. These analyses revealed that alcohol exposure markedly increased the number of TUNEL-positive cells compared with that in liver sections from control mice (Fig. 3). Again, CopA3 treatment significantly attenuated this ethanol-induced effect (Fig. 3) without affecting hepatocyte apoptosis in the absence of ethanol treatment (Fig. 3). These results strongly suggest an anti-apoptotic effect of CopA3.

Figure 3.CopA3 inhibits ethanol-induced hepatic cell apoptosis. (A-B) Mice in the experimental group (n = 6) were pretreated with CopA3 (30 mg/kg, i.p.) and after 1 h were treated with ethanol (EtOH; 3.0 g/kg, i.p.) for 24 h. Controls included mice injected with 0.9% saline only (saline), saline + EtOH (EtOH) or saline + CopA3 (CopA3). (A) Representative images of TUNEL-stained liver tissues prepared from the indicated groups (magnification, 200×). (B) Quantification of apoptotic cells in the H&E-stained sections and expressed as the ratio of TUNEL positive cells to hepatocytes counted per field. Results are presented as means ± SEM (*, p < 0.05).

CopA3 protects the liver against ethanol-induced hepatocyte injury in mice

A key feature of ALD patient livers is a potentially progressive histological change termed hepatocellular ballooning [33, 34], which is characterized by the swelling of liver cells with rarefied cytoplasm generally considered a form of apoptosis [35]. Therefore, quantification of the degree of staining for ballooning degeneration can be helpful for assessing the severity of ALD [33−35]. Applying this concept, we examined whether inhibition of liver cell apoptosis and liver inflammation by CopA3 is associated with changes in ethanol-induced ballooning degeneration in the liver. As shown in Fig. 4A, alcohol exposure markedly increased ballooning degeneration of hepatocytes in mice compared with that in control mice administered saline. Notably, CopA3 administration significantly reduced ballooning degeneration of hepatocytes in livers of ethanol-injected mice (Fig. 4A). Quantification of the extent of ballooning degeneration also confirmed the ability of CopA3 to protect against ethanol-induced liver cell injury (Fig. 4B). Collectively, these data suggest that CopA3 can inhibit mouse liver inflammation and subsequent liver cell injury caused by ethanol toxicity. These results also suggest that the insect-derived antimicrobial peptide CopA3 could be used for treating ethanol-induced liver disease.

Figure 4.CopA3 ameliorates ethanol-induced hepatic ballooning degeneration in mouse livers. Mice in the experimental group (n = 6) were pretreated with CopA3 (30 mg/kg, i.p.) and after 1 h were treated with ethanol (EtOH; 3.0 g/kg, i.p.) for 24 h. Controls included mice injected with 0.9% saline only (saline), saline + EtOH (EtOH) or saline + CopA3 (CopA3). (A) Representative H&E-stained liver sections from experimental mice (magnification, 200×, or 400×). The presented results are representative of three independent experiments. (B) Ballooning degeneration was assessed by histopathological analysis (*, p = 0.064, ethanol-treated group vs. ethanol+CopA3 treatment group).

Acknowledgment

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This work was supported by the Daejin University Research Grants in 2021.

Conflict of Interest

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The authors have no financial conflicts of interest to declare.

References

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