Novel effect of methionine enkephalin against influenza A virus infection through inhibiting TLR7-MyD88-TRAF6-NF-κB p65 signaling pathway

Novel effect of methionine enkephalin against influenza A virus infection through inhibiting TLR7-MyD88-TRAF6-NF-κB p65 signaling pathway

International Immunopharmacology 55 (2018) 38–48 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.el...

2MB Sizes 0 Downloads 19 Views

International Immunopharmacology 55 (2018) 38–48

Contents lists available at ScienceDirect

International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Novel effect of methionine enkephalin against influenza A virus infection through inhibiting TLR7-MyD88-TRAF6-NF-κB p65 signaling pathway

T

Jing Tiana, Xue Jiaob, Xiaonan Wanga, Jin Gengc, Reizhe Wangd, Ning Liue, Xinhua Gaof, ⁎ Noreen Griffing, Fengping Shana, a

Department of Immunology, School of Basic Medical Science, China Medical University, Shenyang 110122, China Department of Translational Medicine, No.4 Teaching Hospital, China Medical University, Shenyang 110032, China c Department of Ophthalmology, No.1 Teaching Hospital, China Medical University, Shenyang 110001, China d Department of Gynecology, No.1 Teaching Hospital, China Medical University, Shenyang 110001, China e Department of Gynecologic Oncology, Shengjing Hospital, China Medical University, Shenyang 110016, China f Department of Dermatology, No.1 Teaching Hospital, China Medical University, Shenyang 110001, China g Immune Therapeutics, Inc., 37 North Orange Avenue, Suite 607, Orlando, FL 32801, USA b

A R T I C L E I N F O

A B S T R A C T

Keywords: Methionine enkephalin Influenza A virus Toll-like receptors Opioid receptors Antiviral efficacy

The morbidity and mortality associated with influenza A virus infections, have stimulated the search for novel prophylactic and therapeutic drugs. The purpose of this study was to investigate the prophylactic and therapeutic effect of synthetic methionine enkephalin (MENK) on mice infected by A/PR/8/34 influenza virus (H1N1) in vivo. The results showed that MENK could exert both prophylactic and therapeutic influences on infected mice, significantly improve the survival rate, relieve acute lung injury and decrease cytokine (IFN-α, IFN-β, TNF-α, IL-6, and IL-1β) levels. MENK also inhibited virus replication on day 4 post infection (p.i.) through upregulating opioid receptors (MOR, DOR) and suppressing TLR7-MyD88-TRAF6-NF-κB p65 signaling pathways. These results suggest that MENK, given via intranasal administration, could provide a novel drug with a new mode of action as a nonspecific anti-influenza agent or vaccine adjuvant.

1. Introduction Influenza A virus (H1N1) is a major respiratory pathogen, but avianorigin influenza viruses, with their high lethality and potential to cause epidemics, is a bigger problem, and has captured the world's attention [1–3]. The mechanism underlying the recurrent epidemics is the evolution of the virus to escape the immunity induced by prior infections and vaccination [4]. The emergence of a new H7N9 avian influenza virus in 2013 has shown the limitation of current vaccines [5]. In addition, the current therapeutic drugs for influenza infection are limited, and some isolated influenza A virus subtypes, are resistant to available drugs [6–9]. Therefore, novel vaccine strategies not affected by viral adaptation or mutation, which would complement the vaccines and antiviral drugs are needed for better influenza control. Although cytokine and chemokine secretion following infection could contribute to the elimination of influenza virus, excessive inflammatory response(s) can also result in serious pathological damage and mortality [10,11]. Studies have shown that an early recruitment of

inflammatory cells, including macrophages, DC, and NK cells, followed by an inflammatory response, is the dominant factor resulting in acute respiratory disease after influenza A virus infection [12]. Toll-like receptors (TLRs) are the mediators in innate and adaptive immunity, controlling infections and activating inflammatory responses against pathogens. TLRs accomplish this by recognizing the molecular patterns specific to microorganisms [13,14]. TLR7 is a nucleotide-sensing TLR, activated by single-stranded RNA. It acts via MyD88 and TRAF, triggering NF-κB p65 activation, cytokine secretion and inflammatory responses [15]. MENK is a naturally occurring endogenous opioid peptide, which is cleaved from pro-enkephalin [16] and found primarily in the adrenal medulla. It has a role in regulating immune and neuroendocrine systems, and modulating functions of cells related to both the innate and adaptive immune systems via binding to opioid receptors [17]. As early as 1995, Burger RA used MENK to treat influenza virus A/NWS/33 (H1N1) infections, observing that MENK had an anti-influenza effect by upregulation of NK and CTL functions [18]. Our team has demonstrated

Abbreviations: DOR, δ-opioid receptors; IAV, influenza A virus; MENK, methionine enkephalin; MOR, μ-opioid receptors; MDCK, Madin-Darby canine kidney; NS, normal saline; p.i., post infection; PR8, influenza strains A/PR/8/34; Rib, ribavirin; TLRs, toll-like receptors ⁎ Corresponding author. E-mail address: [email protected] (F. Shan). https://doi.org/10.1016/j.intimp.2017.12.001 Received 12 October 2017; Received in revised form 29 November 2017; Accepted 1 December 2017 1567-5769/ © 2017 Published by Elsevier B.V.

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

liquid nitrogen for hemagglutination test (HA), and total RNA extract and protein were kept for Western blot analysis. The tissue sections were immune-stained using streptavidin-biotinhorseradish peroxidase. The sections were de-paraffinized, rehydrated and then underwent antigen retrieval using citrate buffer (pH 6.0) at 98 °C for 20 min to unmask antigen epitopes. The sections were treated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase, incubated with 100 μl goat serum for 30 min at 37 °C, and then incubated with antibody MOR(1:200) (Novusbio, cat.no. NB100-1620)/ DOR(1:100) (Abcam, cat.no. ab63536)/TLR7(1:50) (Abcam, cat.no. ab45371)/MyD88(1:200) (Abcam, cat.no. ab 2068)/TRAF6(1:100) (Abcam,cat.no. ab13853)/NF-κB p65 (1:500) (Abcam, cat.no. ab16502) overnight at 4 °C. The slides were rinsed with PBS and incubated with HRP-labeled secondary antibody (zsbio, cat.no. PV9001) at 37 °C for 20 min, DAB staining and then hematoxylin counterstain, dehydrated, and mounted. Stained cells were calculated by the number of positive pixels per area in 3 locations of each slide by Image J 1.42 software (National Institutes of Health, Bethesda, MD).

that MENK stimulates TLR4-Myd88-mediated signal transduction in DC cells. In light of these and other previous findings, we hypothesize that MENK has antiviral activity by inhibiting inflammatory responses following binding to opioid receptors. TLR7-MyD88-TRAF6-NF-κB p65 signaling pathways may be the potential targets for MENK's mechanism of anti-influenza virus (H1N1; PR8) activity. Therefore, the focus of these studies was to elucidate the mechanisms of action by MENK. 2. Materials and methods 2.1. Mice and virus Female C57BL/6 mice (6–8 w) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in specific pathogen-free conditions in accordance with the guide for the Care and Use of Laboratory Animals (NIH Publication) and approved by Animal Use and Care Committee of China Medical University. The mouse-adapted influenza strain A/PR/8/34 (H1N1; PR8) was kindly provided by China Center for Disease Control and Prevention (Beijing, China). The virus was amplified in 10 d-old embryo chicken eggs as described previously [19].

2.5. Hemagglutinin (HA) test Lung tissue was homogenized to a 10% (w/v) suspension with sterilized PBS, and centrifuged at 12,000 × g for 10 min. The supernatant was serially diluted two fold with PBS, 50 μl added to each Ubottom well, 50 μl of 1% guinea pig red blood cells, then mixed and incubated at room temperature for 30 min. The final dilution that completely agglutinated red blood cells was considered the end-point of titration.

2.2. Reagents MENK (≥ 99% purity) was provided by America peptide. Inc. Ribavirin was purchased from Jinan Limin Pharmaceutical Co. Ltd. RNeasy mini kit was purchased from Qiagen. One Step SYBR® Prime Script™ RT-PCR Kit was purchased from TaKaRa. The mAbs of DOR (cat.no. ab63536), TLR7 (cat.no. ab45371), MyD88 (cat.no. ab 2068), TRAF6 (cat.no. ab13853), NF-κB p65 (cat.no. ab16502), β-actin (cat.no. ab16502) were purchased from Abcam, MOR (cat.no. NB1001620) were purchased from Novusbio. Other chemicals frequently used in our laboratory were all from Sigma or Aldrich.

2.6. RNA isolation and qPCR analysis RNA was extracted from lung homogenates (200 μl) and RNA isolated and purified using RNeasy (Qiagen, cat.no. 74104), according to manufacturer's protocol. qPCR was performed using One Step SYBR Prime Script RT-PCR Kit (TaKaRa, cat.no. RRO66A) with QuantStudio 6 Flex Real-time PCR system (ABI). qPCR reactions completed as follows: 5 min at 42 °C and 10 s at 95 °C, followed by 40 cycles-3 s at 95 °C, 30 s at 60 °C-and a melting curve step. Primer sequences are detailed in Table 1. Gene expression was quantified and normalized to GAPDH RNA expression using the 2-△△CT method [20].

2.3. Infection and treatment of mice Female C57BL/6 were assigned to five groups (12 mice/group): normal saline (NS) group, A/PR/8/34 influenza virus (PR8) model control, pre-MENK group, MENK treated group and ribavirin (Rib) treated group. Fig. 1 illustrates an outline of the mouse model. All mice were monitored daily for weight loss and survival for 14 consecutive days post infection (p.i.). Body weight loss > 30% was considered as the critical limit of experiment.

2.7. Western blot Lung tissue was homogenized with lysis buffer containing 1 mM PMSF, 10 μg/ml aprotinin and 10 μg/ml leupeptin. Equal amounts of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane (120 mA, 120 min). Transferred proteins were incubated overnight with specific antibodies against TLR7(1:200)(Abcam, cat.no. ab45371)/MyD88(1:1000) (Abcam, cat.no. ab 2068)/TRAF6(1:500) (Abcam, cat.no. ab13853)/NF-κB p65(1:2000)(Abcam, cat.no.

2.4. Histological examination and immunohistochemistry On day 4 p.i., the lungs of mice in each group were collected. The left lobes were fixed in neutrally buffered 4% formaldehyde and then dehydrated, embedded in paraffin, and cut into 4 μm-thick sections stained with hematoxylin-eosin (HE). The right lobes were stored in

Fig. 1. Experimental design. Female C57BL/6 mice were anesthetized and infected via intranasal instillation (i.n.) with 10 LD50 of influenza A/PR/8/34 H1N1 virus (d0) except mice in NS group. The mice in pre-MENK group were treated via i.n. with 20 mg/kg MENK daily for 6 successive days (d-5-d0) prior to infection. The mice in MENK group were treated via i.n. with 20 mg/kg MENK daily for 7 successive days post infection (d0–d6 p.i.). The mice in normal control group were treated with equal volume of saline for successive 7 days, d0–d6 p.i.) and The mice in positive control group took Ribavirin (100 mg/kg/d for 7 days, d0–d6 p.i.) orally.

39

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

influenza virus challenge, mice were given 10 LD50 viral challenge (Fig. 1). The mice in the PR8 group showed a rapid weight loss from day 2 p.i., while the Pre-MENK and Rib treated mice showed comparative weight loss from day 2 to day 7 p.i. followed by a steady weight gain (Fig. 2b). The survival curves showed that all mice in PR8 group died by day 9 p.i. The pre-MENK treatment significantly prolonged survival to 50.0% (P < 0.01 versus PR8 group). Similarly MENK treatment also improved the mice survival to 33.3% (P < 0.05 versus PR8 group), while Rib treatment improved the mice survival to 83% (P < 0.01 versus PR8 group) (Fig. 2a). There were no difference between preMENK and Rib treatment (P = 0.0567), and significant difference between MENK and Rib treatment (P < 0.01). This suggested that Prophylactic administration of MENK and Rib treatment had similar therapeutic effects on survival of mice challenged with PR8 virus. Also survival rate in pre-MENK group was higher than that in MENK group (P < 0.05). The results demonstrated that prophylactic or therapeutic administration of MENK ameliorated lethal influenza infection, and prophylactic efficacy of MENK was much better than its therapeutic effect.

Table 1 PCR primer sequences. Primer

Direction

Sequence

Virus

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

GACCGATCCTGTCACCTCTGAC-3′ AGGGCATTCTGGACAAAGCGTCTA-3′ 5′-ATGGCTAGGCTCTGTGCTTT-3′ 5′-CTCTTGTTCCTGAGGTTAT-3′ 5′-AGCTCCAAGAAAGGACGAACAT-3′ 5′-GCCCTGTAGGTGAGGTTGATCT-3′ 5′-CCAAAGGGATGAGAAGTTCC-3′ 5′-CTCCACTTGGTG GTTTGCTA-3′ 5′-ATGAAGTTCCTCTCTGCAAG-3′ 5′-GTGTAATTAAGCCTCCGACT-3′ 5′-TAGGCTCATCTGGGATCCTC-3′ 5′-AAAAGGTGGCATTTCACAGT-3′ 5′-CCCAGTTCTTTATGCGTTCCT-3′ 5′-ATTAGCCGTGGAGGGGTGT-3′ 5′-ATGTAAAGAGGGCTGGGAATGTAG-3′ 5′-GGGTTGGTTTTGTTGTTTGGA-3′ 5′-GGTGGCAAAATTGGAAGATCC-3′ 5′-AGCTGTATGCTCTGGGAAAGGTT-3′ 5′-TGGTGGTTGTTTCTGACGAT-3′ 5′-GGAAAGTCCTTCTTCATCGC-3′ 5′-TCCAGGGACTGGTTTAACCC-3′ 5′-GCCGCCTGGAGCATATTTCA-3′ 5′-ATGTGCATCGGCAAGTGG-3′ 5′-CAGAAGTTGAGTTTCGGGTAG-3′ 5′-ACCACCATGGAGAAGGCTGG-3′ 5′-CTCAGTGTAGCCCAGGATGC-3′

IFN-α IFN-β TNF-α IL-6 IL-1β MOR DOR TLR7 MyD88 TRAF NF-κB p65 GAPDH

3.2. MENK administration alleviated acute lung injury The main pathological damage of PR8 virus infection was viral interstitial pneumonia that occurred day 4 p.i. Lungs were collected on day 4 p.i. for gross observation and histopathological examination. Our results showed extensive hemorrhagic/inflammatory areas in mice infected with PR8 virus. In comparison, less lung lesion occurred in mice treated with MENK, while more restricted damage was observed in the pre-MENK and Rib treatment groups. No pathology was observed in mice in the control group (Fig. 3a). Histopathological examination of mice in the PR8 group revealed extensive parenchymal and peribronchiolar inflammation, epithelial necrosis of airways, diffused alveolar damage with inflammatory cells infiltration, edema, and hemorrhage. Compared to PR8 group, pre-MENK, MENK and Rib treatment reduced the pathology, lesion rates accounted for 23.22%, 44.59%, 11.67% respectively (P < 0.01). And pre-MENK administration significantly decreased the number of influenza-related focal lesions and lung consolidation compared to that in mice with MENK treatment (P < 0.01) (Fig. 3b).

ab16502)/β-actin(1:5000) (Abcam, cat.no. ab 8226). After washing, the blots were incubated with a secondary antibody (1:10,000) (EarthOx, cat.no. E030120-01). Signals were detected by luminol chemiluminescence and quantified by computer-assisted image analysis. The data was normalized to β-actin and expressed as fold increase versus NS group. 2.8. Statistical analyses All results are presented as mean ± SEM. For comparison of > 2 groups, one-way ANOVA, followed by Tukey post-test was used. Statistical analysis was performed using GraphPad Prism 5. A p value < 0.05 was considered statistically significant (★P < 0.05, ★★ P < 0.01).

3.3. MENK administration decreased influenza replication in mice The viral titers of the lungs of mice infected with PR8 virus reached significantly higher levels at day 4 p.i., compared to the NS group (P < 0.01), the pre-MENK group (P < 0.01), and the MENK and (P < 0.05) Rib treatment group (P < 0.01) (Fig. 4a). The relative viral amplification was markedly higher (14,146-fold) on day 4 p.i. in the PR8 group, while 3943-fold in pre-MENK group, 6638-fold in MENK

3. Results 3.1. MENK increased survival of mice challenged with PR8 virus To assess the prophylactic and therapeutic efficacy of MENK on

Fig. 2. The influence of MENK on the mice infected with Flu. A. Prophylactic and therapeutic administration of MENK ameliorated lethal influenza A/PR/8/34 H1N1virus challenge. The survival rate (a) and weight change (b) in each group of 12 mice was monitored daily for 14 days. Data were analyzed using the log-rank (Mantel-Cox) test. ★P < 0.05, ★★P < 0.01 versus the influenza A/PR/8/34 H1N1 virus group.

40

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

Fig. 3. MENK administration improved acute lung injury in mice challenged with 10 LD50 influenza A virus (PR8). Lungs of mice infected with influenza virus were collected at day 4 p.i. (a) Macroscopic changes of appearance of pulmonary tissue; (b) histopathological changes in lung tissues (HE). No histopathologic lesion in normal control; In PR8 group(virus infection control): extensive epithelial necrosis of bronchioles with immune cell infiltration (black arrow), diffuse alveolar damage with intra-alveolar edema, hemorrhage, inflammatory cells infiltration (white arrow), thickened alveolar wall (★), and large areas of parenchymal inflammation (☆). In pre-MENK group (mice treated with MENK prior to virus infection): mild bronchiolar inflammation (black arrow), a few alveolar damage with hemorrhage, inflammatory cells infiltration (white arrow). In MENK treated group(mice infected with virus, followed by treatment with MENK): moderate immune cell infiltration around bronchioles (black arrow) and alveolar (white arrow), a number of alveolar wall thickened with inflammatory exudation(★). In Rib group (mice infected with virus, followed by treatment with Ribavirin): slight bronchiolar (black arrow) and alveolar (white arrow) inflammation; (c) panel were presented as rate over the PR8 group. Data represent the mean ± SEM of three independent experiments. One-way ANOVA with the Tukey-Kramer posttest was performed: ★ P < 0.05, ★★P < 0.01 versus the influenza A/PR/8/34 H1N1virus group. Scale Bar: 100 μm.

inhibit the secretion of inflammatory cytokines as shown in Fig. 5a. High levels of pulmonary antiviral and inflammatory cytokines were induced on day 4 post-infection with the PR8 virus including: IFN-α, 8.1-fold; IFN-β, 76.8-fold; TNF-α, 140.6-fold; IL-6, 347.8-fold; and IL1β, 14.0-fold. In contrast, the administration of MENK prior to infection (pre-MENK) decreased transcription of the above cytokines in the lungs of infected mice (IFN-α, 3.6-fold; IFN-β, 26.2-fold; TNF-α, 47.1-fold; IL6, 90.4-fold; and IL-1β, 4.1-fold) compared to the lungs of uninfected mice (P < 0.01). Correspondingly, the therapeutic effect of MENK yielded reductions in cytokine expression (IFN-α, 6.5-fold; IFN-β, 49.3fold; TNF-α, 83.4-fold; 167.1-fold; and IL-1β, 8.1-fold) compared with

group and 1675-fold in Rib group (Fig. 4b) compared with that in the NS group. These findings suggested that both prophylactic and therapeutic administration of MENK could reduce the replication of influenza virus in mice effectively, but that prophylaxis was more effective.

3.4. MENK administration reduces inflammatory response in mice Challenge with influenza H1N1 PR8 strain is characterized by a robust inflammation. It is more significant in avian influenza infections, resulting in morbidity and pathogenesis as a consequence of the induced inflammatory response. We observed that MENK treatment could 41

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

Fig. 4. MENK administration inhibited virus replication. (a) HA titers of lung homogenates. Lungs were collected at day 4 p.i. and the supernatants of homogenized lung were determined for virus titers. (b) Virus M gene expressions in the lung cells of mice in all groups were quantified at d 4 p.i. by quantitative PCR (qPCR). Results were presented as fold increase over the NS group. Data represent the mean ± SEM of three independent experiments. One-way ANOVA with the Tukey-Kramer posttest was performed: ★P < 0.05, ★★P < 0.01 versus influenza A/PR/8/34 H1N1 virus group.

Fig. 5. MENK regulated production of inflammatory cytokines in the lungs cells of mice challenged with 10 LD50 influenza A virus (PR8). quantitative PCR (qPCR) was performed to confirm gene expressions of inflammatory cytokines (IFN-α, IFN-β, TNF-α, IL-6, and IL-1β) in the lungs cells of mice in all groups at d 4 p.i. Results were presented as fold increase over the NS group. Data represent the mean ± SEM of three independent experiments. One-way ANOVA with the Tukey-Kramer posttest was performed: ★P < 0.05, ★★P < 0.01 versus influenza A/PR/8/34 H1N1virus group.

As shown in Fig. 6b, c, MOR and DOR were mainly focused on epithelial and mesenchymal cells, and inflammatory cells. The level of MOR and DOR yielded 4.1, 2.4 × 105 pixel/area in MENK treated mice and 3.5, 1.8 × 105 in pre-MENK treated mice, which was significantly higher than that in PR8 mice (P < 0.01) as shown in Fig. 6d. While MOR or DOR was not changed in PR8 and Rib treatment mice compared with the saline-treated mice (P > 0.05). We conclude that MENK upregulated the expressions of MOR and DOR receptors and suggest that MENK has as part of it mechanism of flu therapy the upregulation of its receptor.

those in lungs of uninfected mice (P < 0.01). Compared with PR8 group, all above cytokines were significantly lower in pre-MENK and MENK group (P < 0.01), except IFN-α in MENK group (P < 0.05). There were significantly different of IFN-α, IFN-β, TNF-α, IL-6, IL-1β between pre-MENK and MENK group (P < 0.01). Note that the inhibiting effect in the pre-MENK group was significantly greater than that in the therapeutic MENK group. These results would further support that MENK can control virus-induced lung inflammation and pathologic damage. 3.5. MENK administration upregulated opioid receptor (MOR and DOR)

3.6. MENK administration downregulated TLR7-MyD88-TRAF6-NF-κB p65 signaling pathway

To assess the mechanistic basis for the effect of MENK on influenza A virus infection, we detected mRNA expressions of DOR and MOR in mice. There were no significant changes compared with the levels measured in mice from the NS, PR8 and Rib group. However there was a marked increase in MOR or DOR in mice in pre-MENK group (MOR, 9.3-fold; DOR, 6.4-fold) and in the MENK group (MOR, 6.1-fold; DOR, 4.8-fold), and had statistical significance in both group (P < 0.05) MOR and DOR were significantly higher in pre-MENK and MENK group than PR8 group (P < 0.01) as shown in Fig. 6a,

It has been proven that inflammatory responses caused by influenza virus infection occur via the activation of the TLRs signaling pathway. Thus, we measured the expressions of TLR7, MyD88, TRAF6, NF-κB p65 in pulmonary tissues on day 4 p.i. As shown in Fig. 7b, the protein expressions of TLRs pathway in mice following PR8 infection significantly were increased such: TLR7, 3.9-fold; MyD88, 5.9-fold; TRAF6, 4.5-fold; and p65, 4.6-fold. However, these increases could be 42

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

Fig. 6. MENK treatment upregulated opioid receptor to defense influenza A virus (PR8). Quantitative PCR (qPCR) was performed to confirm gene expressions of opioid receptor (MOR and DOR) in the lungs cells of mice in all groups at d 4 p.i (a). Results were presented as fold increase over the NS group. Immuno-histochemical staining of cellular localization (dark brown) of MOR (b), DOR (c) was mainly expressed performed on cell membrane as multi pass membrane protein. Generally, MOR and DOR were mainly focused on epithelial and mesenchymal cells, and inflammatory cells. In MENK treatment group, there were extensive and intense MOR and DOR staining than other groups. (d) Panel represents quantified data. Data represent the mean ± SEM of three independent experiments. One-way ANOVA with the Tukey-Kramer posttest was performed: ★P < 0.05, ★★P < 0.01 versus the influenza A/ PR/8/34 H1N1virus group. Scale Bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

43

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

Fig. 6. (continued)

destruction. MENK's anti-viral activity is associated with the inhibition of inflammatory responses by down-regulating TLR7-MyD88-TRAF6NF-κB p65 signaling pathways. Moreover, as expected based on disease burden, the effect of prophylactic administration was significantly greated than that of therapeutic administration. It has been clear that broad inflammatory response and several factors of innate immune system act as the main players on morbidity and pathogenesis caused by H1N1 infection. Type I IFN (IFNα/β) is considered a critical mediator of virus clearance during the period of influenza virus infection [44]. Immoderate IFN (IFNα/β) has a pernicious impact on disease severity, mostly leading to damaging effects on the host, such as excessive pro-inflammatory cytokines, inflammatory cells infiltration, and oxidative stress and necrocytosis [45]. TNF-α has multiple immune effects, such as stimulation of inflammatory responses, cell proliferation, cell differentiation, and induction of cell apoptosis [46]. Based on the multiple effects of TNF-α in tissue inflammation and pathological injury, it is crucial to mount immunotherapy. Shi X et al. found that mice challenged with influenza virus by using etanercept, one of TNF α-blocking agents, suppressed immunopathology and morbidity [47]. Uncontrolled expression of IL6 has been related to several pathological manifestations, and extensively studied as a therapeutic target [48]. Several reports have shown that IL1β has critical roles in tissue inflammatory injury in mouse models [49], and numerous production of IL1β would cause acute lung tissue injury in the case of severe influenza infection. Therefore, anti-inflammatory therapy was as an evidence of principle in alleviating IAV pathology. Our data showed that MENK can significantly reduce the immoderate expression of IFN-α, IFN-β, TNF-α, IL-6, IL-β, and alleviate the pathological damage of lung tissue caused by the imbalance of inflammatory mediators. The opioid receptors (MOR, DOR) were upregulated by MENK administration, while not changed in PR8 and Rib treatment mice compared with the saline-treated mice. We also found that the proportion of receptor upregulation was associated with the degree of inhibition of inflammatory cytokines. The degree of upregulation of receptors and inhibition of inflammatory cytokines in prophylactic administration of MENK was higher versus when MENK works as therapeutic administration. This indicated that MENK played an anti-influenza effect via binding to opioid receptors, as expressed on epithelial and mesenchymal cells, and immune cells of lung in mice. MENK, while in prophylactic administration, up-regulated the status of lung cells and activated innate immune cells such as NK, DC, and Monocytes before infection. Once the influenza virus invaded the host, immune cells were quickly recruited to the infected site to clear the influenza virus, which may be the main reason for better preventive effect than that of therapeutic administration. It should be noted that as previous experimental results showed MENK could effectively reduce the titer of influenza B virus in the chicken embryo experiment, it had no direct effect on anti-influenza B virus on MDCK cells infection. Altogether, we demonstrated the antiviral effect of MENK was targeted toward innate immune response of host via opioid receptors, and not toward the virus itself. Having established that MENK administration provides some

inhibited by Pre-MENK treatment (TLR7, 1.7-fold; MyD88, 1.4-fold; TRAF6, 1.6-fold and p65, 1.9-fold) (P < 0.01), MENK treatment (TLR7, 2.2-fold; MyD88, 3.4-fold; TRAF6, 2.4-fold and p65, 2.6-fold) (P < 0.01), or Rib treatment (TLR7, 1.3-fold; MyD88, 1.5-fold; TRAF6, 1.2-fold and p65, 1.3-fold) (P < 0.01). mRNA analysis by qPCR further confirmed changes in the expression of TLR7/NF-κB p65 in mice infected with influenza virus and treated with MENK. As shown in Fig. 7c, the mRNA expressions of TLR7, MyD88, TRAF6 and NF-κB p65 were significantly increased in PR8 group (8.2- to 17.1-fold), which was significantly reduced by treatment with pre-MENK(3.3- to 7.6-fold), MENK(5.8- to 11.8-fold) or Rib(1.5- to 4.1-fold). The trend toward the inhibition of inflammatory cytokines was similar to the data from Western blotting. As shown in Fig. 7d–g, Immuno-histochemical staining of immune-positive cells were located in inflammatory cells, bronchial epithelial cells, interstitial cells and pneumocytes. The number of positive cells and intensity of immunoreaction within lungs indicated that expressions of TLR7, MyD88 in mice from the pre-MENK group (2.7, 3.5 pixel/area), MENK group (3.8, 5.3 pixel/area), and Rib treatment group (2.2, 2.8 pixel/ area) were less intense than the PR8 virus infected mice (5.5, 10.8 pixel/area) (P < 0.01). In comparison to the PR8 group, the level of TRAF6 and p65 yielded 2.9, 2.7 × 105 pixel/area in MENK group (P < 0.05), 4.6, 5.5 × 105 in Pre-MENK group (P < 0.01), and 2.1, 2.2 × 105 in Rib group (P < 0.01). MENK and Rib administration significantly down-regulated TLR7, MyD88, TRAF6 and p65 activity (Fig. 7h). 4. Discussion Vaccination is the most effective and economical way to protect against influenza infections. The pathogenicity of influenza virus is associated with inflammatory responses, contributing to acute pulmonary injury that can be fatal [21–23]. Therefore, regulating the inflammatory responses caused by influenza virus infection, rather or in addition to the influenza virus itself provides a potential strategy as an antiviral. MENK, as immune regulatory factor, can control the expansion and activation of natural killer cells [24,25], DC [26–28], macrophages [29,30], CD4 +T cells [31,32], and CD8 + T cells [33] via opioid receptor (MOR, DOR) expression on immune cells, MENK also regulated NK cells, macrophages, dendritic cells, T cells [34], epithelial and mesenchymal cells [35], and hepatocellular [36], and melanoma cells [37]. Appropriate concentration of MENK has shown significant immunotherapeutic effect on tumors, including pancreatic, breast, ovarian, hepatoblastoma, and kidney cancers [38–41]. Moreover, MENK has potential antiviral activity including hepatitis [42], HIV [43], and influenza virual infections [18]. The present study demonstrated that prophylactic and therapeutic administration of MENK by intranasal administration significantly improved survival, reduced weight loss, and acute pulmonary injury during H1N1 PR8 virus infection. While regulation of innate immunity has as its goal to enhance immune responses to clear viral infections, there are also negative consequences that might induce tissue 44

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

(caption on next page)

45

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

Fig. 7. MENK inhibited the NF-κB signaling pathway in mice challenged with 10 LD50 influenza A virus (PR8). Lungs were collected at day 4 p.i. (a and b) and Western blot was used for confirmation of changes of TLR7, MyD88, TRAF6, and p65. The results were presented as fold increase over the NS group. Gene expressions of TLR7, MyD88, TRAF6, and p65 were quantified at d 4 p.i. by quantitative PCR in the lungs cell of mice in all groups (c). The results were presented as fold increase over the NS group. Immuno-histochemical staining of cellular localization (dark brown) of TLR7 (d), MyD88 (e), TRAF6 (f), and NF-κB p65 (g) were performed: TLR7 (endoplasmic reticulum membrane, endosome, lysosome), MyD88 (cytoplasm), TRAF6/NF-κB p65 (cytoplasm, nucleus). TLR7, MyD88, TRAF6, and p65 in normal mice lungs cells, were mainly expressed within interstitial cells. Generally, MyD88 expression was more than others. In PR8 infected mice lungs cells, there were extensive and intense TLR7, MyD88, TRAF6, and p65 staining within inflammatory cells, bronchial epithelial cells, interstitial cells and pneumocytes. In MENK treated group TLR7, MyD88, TRAF6, and p65 staining of cells mainly focused on inflammatory cells and interstitial cells, with moderate brown intensity. In Pre-MENK and Rib groups, TLR7, MyD88, TRAF6, and p65 staining of cells was located within inflammatory cells and interstitial cells, but with less immune-positive cells and lower intensity of immunoreactions. (h) Panel represents quantified data. Data represent the mean ± SEM of three independent experiments. One-way ANOVA with the Tukey-Kramer posttest was performed: ★P < 0.05, ★★P < 0.01 versus the influenza A/PR/8/34 H1N1virus group. Scale Bar: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. (continued)

46

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

protection against lethal H1N1 PR8 virus infection, we followed the search in which signaling pathway was triggered by MENK in response to influenza virus infection. When the pathogen invaded, the innate immune recognition was made mainly through identifying the pathogen composition (PAMP), and by matching PRR on the host immune cell [50]. TLRs are important receptors of PRRs, highly expressed on immune cells such as DCs, macrophages, and several T cell subsets, and other cells such as lung cells [51,52]. TLR7 is one of the members of TLR family, located in the endosomal compartments and binds to nucleic acids [53], which are natural barriers against ssRNA viruses through stimulating the downstream signal protein of MyD88. MyD88 is an intercellular adapter protein of multiple innate immune receptors, recruiting and phosphorylating IRAK, and then binding with TRAF. This leads toa series of reactions that active the NF-κB signaling pathway, causing production of proinflammatory cytokines and chemokines, which can induce innate and adaptive immunity to pathogens [54]. The experimental results demonstrated that expression of TLR7, MyD88, TRAF6, NF-κB p65 following MENK administration were significantly reduced compared with those in PR8 treated mice. Furthermore, combined with the results from the studies examining the inflammatory cytokines, we suggest that MENK inhibition of innate immune responses occurs through the down regulation of TLR7MyD88-dependent NF-κBp65 signaling pathway, leading to decreased type I interferon and inflammatory cytokines. Residual inflammatory cytokines would contribute to clearing the PR8 virus and prolonging survival resulting in a protective role of anti-virus infection. In summary, our result sound that (1) MENK can significantly reduce weight loss and prolong survival in mice challenged with PR8 virus. (2) MENK can reduce acute lung injury and decrease viral titers and relative viral amplification. (3) MENK can inhibit uncontrolled innate immune responses, reduce secretion of inflammatory cytokines, such as IFN-α, IFN-β, TNF-α, IL-6 and IL-1β, through down-regulating the TLR7-MyD88-dependent NF-κB p65 signaling pathway. (4) Both prophylactic and therapeutic administration of MENK has anti-viral efficacy, but the former is more effective. MENK targets an innate immune response via the host by opioid receptors, and not toward the virus itself. In regard to this mechanism, it is likely to be effective against all strains of influenza virus. Therefore, we may consider MENK as a potential adjuvant to be used in vaccine preparations against viral diseases like influenza virus infection. The intranasal instillation will make MENK as a more reliable and valuable candidate for mucosal immune vaccine. Further studies are necessary to pursue these initial observations, such as how the innate immune cells, involved in regulatory effects of MENK, could interact with each other, and how MENK would modulate adaptive immune response to play anti-influenza effect.

[2] V.A. Meliopoulos, E.A. Karlsson, L. Kercher, T. Cline, P. Freiden, S. Duan, et al., Human H7N9 and H5N1 influenza viruses differ in induction of cytokines and tissue tropism, J. Virol. 88 (22) (2014) 12982–12991. [3] J.R. Plourde, J.A. Pyles, R.C. Layton, S.E. Vaughan, J.L. Tipper, K.S. Harrod, Neurovirulence of H5N1 infection in ferrets is mediated by multifocal replication in distinct permissive neuronal cell regions, PLoS One 7 (10) (2012) e46605. [4] N. Wibowo, F.K. Hughes, E.J. Fairmaid, L.H. Lua, L.E. Brown, A.P. Middelberg, Protective efficacy of a bacterially produced modular capsomere presenting M2e from influenza: extending the potential of broadly cross-protecting epitopes, Vaccine 32 (29) (2014) 3651–3655. [5] J. Zhou, D. Wang, R. Gao, B. Zhao, J. Song, X. Qi, et al., Biological features of novel avian influenza A (H7N9) virus, Nature 499 (7459) (2013) 500–503. [6] S.I. van de Wakker, M.J.E. Fischer, R.S. Oosting, New drug-strategies to tackle viralhost interactions for the treatment of influenza virus infections, Eur. J. Pharmacol. 809 (2017) 178–190. [7] C.W. Seibert, S. Rahmat, F. Krammer, P. Palese, N.M. Bouvier, Efficient transmission of pandemic H1N1 influenza viruses with high level oseltamivir resistance, J. Virol. 86 (2012) 5386–5389. [8] A.C.L.S. Hurt, D.J. Speers, I.G. Barr, S. Maurer-Stroh, Mutations I117V and I117M and oseltamivir sensitivity of pandemic (H1N1) 2009 viruses, Emerg. Infect. Dis. 18 (2012) 109–112. [9] H. Chen, C.L. Cheung, H. Tai, P. Zhao, J.F. Chan, V.C. Cheng, et al., Oseltamivirresistant influenza A pandemic (H1N1) 2009 virus, Hong Kong, China, Emerg. Infect. Dis. 15 (2009) 1970–1972. [10] L.P. Tavares, M.M. Teixeira, C.C. Garcia, The inflammatory response triggered by influenza virus: a two edged sword, Inflamm. Res. 66 (4) (2017) 283–302. [11] D. Kobasa, S.M. Jones, K. Shinya, J.C. Kash, J. Copps, H. Ebihara, et al., Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus, Nature 445 (7125) (2007) 319–323. [12] N.L. La Gruta, K. Kedzierska, J. Stambas, P.C. Doherty, A question of self-preservation: immunopathology in influenza virus infection, Immunol. Cell Biol. 85 (2) (2007) 85–92. [13] D. Das, I. Sengupta, N. Sarkar, A. Pal, D. Saha, M. Bandopadhyay, et al., Anti-hepatitis B virus (HBV) response of imiquimod based toll like receptor 7 ligand in hbvpositive human hepatocellular carcinoma cell line, BMC Infect. Dis. 17 (1) (2017) 76. [14] G. Harris, R. KuoLee, W. Chen, Role of toll-like receptors in health and diseases of gastrointestinal tract, World J. Gastroenterol. 12 (2006) 2149–2160. [15] Y. Hu, X. Cong, L. Chen, J. Qi, X. Wu, M. Zhou, et al., Synergy of TLR3 and 7 ligands significantly enhances function of DCs to present inactivated PRRSV antigen through TRIF/MyD88-NF-κB signaling pathway, Sci. Rep. 6 (2016) 23977. [16] I. Marcotte, E.J. Dufourc, M. Ouellet, M. Auger, Interaction of the neuropeptide met-enkephalin with zwitterionic and negatively charged bicelles as viewed by 31P and 2H solid state NMR, Biophys. J. 85 (2003) 328–339. [17] M. Piva, J.I. Moreno, F.S. Jenkins, J.K. Smith, J.L. Thomas, C. Montgomery, et al., In vitro modulation of cytokine expression by enkephalin-derived peptide, Neuroimmuno-modulation, vol. 12, 2005, pp. 339–347. [18] R.A. Burger, R.P. Warren, J.H. Huffman, R.W. Sidwell, Effect of methionine enkephalin on natural killer cell and cytotoxic T lymphocyte activityin mice infected with influenza A virus, Immunopharmacol. Immunotoxicol. 17 (2) (1995) 323–334. [19] A. Kawaguchi, T. Suzuki, Y. Ohara, K. Takahashi, Y. Sato, A. Ainai, et al., Impacts of allergic airway inflammation on lung pathology in a mouse model of influenza A virus infection, PLoS One 12 (2) (2017) e0173008. [20] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) method, Methods 25 (2001) 402–408. [21] S. Tripathi, M.R. White, K.L. Hartshorn, The amazing innate immune response to influenza A virus infection, Innate Immun. 21 (1) (2015) 73–98. [22] J.R. Teijaro, The role of cytokine responses during influenza virus pathogenesis and potential therapeutic options, Curr. Top. Microbiol. Immunol. 386 (2015) 3–22. [23] R.V. D'Elia, K. Harrison, P.C. Oyston, R.A. Lukaszewski, G.C. Clark, Targeting the “cytokine storm” for therapeutic benefit, Clin. Vaccine Immunol. 20 (3) (2013) 319–327. [24] J. Kowalski, D. Belowski, J. Wielgus, Bidirectional modulation of mouse natural killer cell and macrophage cytotoxic activities by enkephalins, Pol. J. Pharmacol. 47 (1995) 327–331. [25] Q. Wang, X. Gao, Z. Yuan, Z. Wang, Y. Meng, Y. Cao, et al., Methionine enkephalin (MENK) improves lymphocyte subpopulations in human peripheral blood of 50 cancer patients by inhibiting regulatory T cells (Tregs), Hum. Vaccin. Immunother. 10 (7) (2014) 1836–1840. [26] V.P. Makarenkova, C. Esche, N.V. Kost, G.V. Shurin, B.S. Rabin, et al., Identification of delta and mu type opioid receptors on human and murine dendritic cells, J. Neuroimmunol. 117 (2001) 68–77. [27] A. Be'nard, J. Boue, E. Chapey, M. Jaume, B. Gomes, et al., Delta opioid receptors mediate chemotaxis in bone marrow-derived dendritic cells, J. Neuroimmunol. 197 (2008) 21–28. [28] W. Li, J. Meng, X. Li, H.M.Y. Hua, Q. Wang, et al., Methionine enkephalin (MENK) improved the functions of bone marrow-derived dendritic cells (BMDCs) loaded with antigen, Hum. Vaccin. Immunother. 8 (9) (2013) 1236–1242. [29] S. Stanojević, K. Mitić, V. Vujić, V. Kovacević-Jovanović, M. Dimitrijević, The influence of stress and methionine enkephalin on macrophage functions in two inbred rat strains, Life Sci. 80 (2007) 901–909. [30] W. Chen, J. Liu, J. Meng, C. Lu, X. Li, E. Wang, F. Shan, Macrophage polarization induced by neuropeptide methionine enkephalin (MENK) promotes tumoricidal responses, Cancer Immunol. Immunother. 61 (10) (2012) 1699–1711. [31] H. Ohmori, K. Fujii, T. Sasahira, Y. Luo, M. Isobe, N. Tatsumoto, H. Kuniyasu,

Conflict of interest We, the authors, formally inform the editorial office that we have no conflict of interest here. Acknowledgements This work was supported financially by National Natural Science Foundation of China (31670921 to Fengping Shan) and China Liaoning Province Supporting Construction of Discipline Platforms in Universities. We thank other people whose names were not mentioned here due to the space limitation. References [1] J. He, Z.W. Liu, Lu YP, T.Y. Li, X.J. Liang, P.C. Arck, et al., A systematic review and meta-analysis of influenza A virus infection during pregnancy associated with an increased risk for stillbirth and low birth weight, Kidney Blood Press. Res. 42 (2) (2017) 232–243.

47

International Immunopharmacology 55 (2018) 38–48

J. Tian et al.

[32]

[33]

[34]

[35]

[36]

[37]

[38] [39]

[40]

[41]

(2013) 1066–1070. [42] R. Martinić, H. Sošić, P. Turčić, P. Konjevoda, A. Fučić, R. Stojković, et al., Hepatoprotective effects of Met-enkephalin on acetaminophen-induced liver lesions in male CBA mice, Molecules 19 (8) (2014) 11833–11845. [43] R. Bowden, S.M. Tate, S. Soto, S. Specter, Alteration of cytokine levels in murine retrovirus infection: modulation by combination therapy, Int. J. Immunopharmacol. 21 (12) (1999) 815–827. [44] U. Muller, U. Steinhoff, L.F. Reis, S. Hemmi, J. Pavlovic, R.M. Zinkernagel, et al., Functional role of type I and type II interferons in antiviral defense, Science 264 (1994) 1918–1921. [45] S. Davidson, S. Crotta, T.M. Mccabe, A. Wack, Pathogenic potential of interferon αβ in acute influenza infection, Nat. Commun. 5 (2014) 3864. [46] S. Sethu, A.J. Melendez, New developments on the TNFα-mediated signaling pathways, Biosci. Rep. 31 (2011) 63–76. [47] X. Shi, W. Zhou, H. Huang, H. Zhu, P. Zhou, H. Zhu, et al., Inhibition of the inflammatory cytokine tumor necrosis factor-alpha with etanercept provides protection against lethal H1N1 influenza infection in mice, Crit. Care 17 (2013) R301. [48] S. Kang, T. Tanaka, T. Kishimoto, Therapeutic uses of anti-interleukin-6 receptor antibody, Int. Immunol. 27 (2015) 21–29. [49] C.A. Dinarello, The IL-1 family and inflammatory diseases, Clin. Exp. Rheumatol. 20 (2002) S1–13. [50] J.H. Kreijtz, R.A. Fouchier, G.F. Rimmelzwaan, Immune responses to influenza virus infection, Virus Res. 162 (2011) 19–30. [51] R. Kulkarni, S. Behboudi, S. Sharif, Insights into the role of toll-like receptors in modulation of T cell responses, Cell Tissue Res. 343 (2011) 141–152. [52] M. Derda, A. Wojtkowiak-Giera, A. Kolasa-Wołosiuk, D. Kosik-Bogacka, E. Hadaś, P.P. Jagodziński, et al., Acanthamoeba infection in lungs of mice expressed by tolllike receptors (TLR2 and TLR4), Exp. Parasitol. 165 (2016) 30–34. [53] T. Kawai, S. Akira, The role of pattern-recognition receptors in innate immunity: update on toll-like receptors, Nat. Immunol. 11 (2010) 373–384. [54] A.L. Blasius, B. Beutler, Intracellular toll-like receptors, Immunity 32 (3) (2010) 305–315.

Methionine-enkephalin secreted by human colorectal cancer cells suppresses T lymphocytes, Cancer Sci. 100 (3) (2009) 497–502. F. Shan, Y. Xia, N. Wang, J. Meng, C. Lu, Y. Meng, N.P. Plotnikoff, Functional modulation of the pathway between dendritic cells (DCs) and CD4+ T cells by the neuropeptide: methionine enkephalin (MENK), Peptides 32 (2011) 929–937. W. Li, W. Chen, H. Ronald, N.P. Plotnikoff, G. Youkilis, N. Griffin, et al., Immunotherapy of cancer via mediation of cytotoxic T lymphocytes by methionine enkephalin (MENK), Cancer Lett. 344 (2) (2014) 212–222. M.J. Finley, C.M. Happel, D.E. Kaminsky, T.J. Rogers, Opioid and nociceptin receptors regulate cytokine and cytokine receptor expression, Cell. Immunol. 252 (2008) 146–154. M.E. Sunday, K.J. Haley, R.L. Emanuel, J.S. Torday, N. Asokananthan, K.A. Sikorski, et al., Fetal alveolar epithelial cells contain [D-Ala(2)]-deltorphin I-like immunoreactivity: delta- and mu-opiate receptors mediate opposite effects in developing lung, Am. J. Respir. Cell Mol. Biol. 25 (4) (2001) 447–456. D.M. Avella, E.T. Kimchi, R.N. Donahue, H.R. Tagaram, P.J. McLaughlin, I.S. Zagon, et al., The opioid growth factor-opioid growth factor receptor axis regulates cell proliferation of human hepatocellular cancer, Am. J. Physiol. Regul. Integr. Comp. Physiol. 298 (2) (2010) R459–66. D.M. Wang, G.C. Wang, J. Yang, N.P. Plotnikoff, N. Griffin, Y.M. Han, et al., Inhibition of the growth of human melanoma cells by methionine enkephalin, Mol. Med. Rep. 14 (6) (2016) 5521–5527. I.S. Zagon, P.J. McLaughlin, Opioid growth factor and the treatment of human pancreatic cancer: a review, World J. Gastroenterol. 20 (9) (2014) 2218–2223. I.S. Zagon, N.K. Porterfield, P.J. McLaughlin, Opioid growth factor-opioid growth factor receptor axis inhibits proliferation of triple negative breast cancer, Exp. Biol. Med. (Maywood) 238 (6) (2013) 589–599. I.S. Zagon, R. Donahue, P.J. McLaughlin, Targeting the opioid growth factor: opioid growth factor receptor axis for treatment of human ovarian cancer, Exp. Biol. Med. (Maywood) 238 (5) (2013) 579–587. M. Rogosnitzky, M.J. Finegold, P.J. McLaughlin, I.S. Zagon, Opioid growth factor (OGF) for hepatoblastoma: a novel non-toxic treatment, Investig. New Drugs 31 (4)

48