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Protective effect of homonojirimycin from Commelina communis (dayflower) on influenza virus infection in mice.

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Keywords:

Commelina communis L.

Homonojirimycin

Influenza virus

Cytokine

ABSTRACT

The effects of homonojirimycin (HNJ), one of alkaloids from Commelina communis L., on protection against influenza virus infection in mice were investigated. HNJ was found to improve the survival rate, prolong the mean survival time and reduce virus yields in lungs on days 4 and 6 post-infection (p.i.), after the agent had been orally administered to the mice from 2 days before infection to 6 days p.i. Administration of HNJ (1 mg/kg) significantly increased interferon (IFN)-[gamma] and interleukin (IL)-10 levels but decreased tumor necrosis factor (TNF)-[alpha] and IL-6 levels in serum and lungs of influenza-infected mice on days 2, 4 or 6 p.i. These results showed that HNJ exerted protection against influenza virus infection and produced effective immune responses in vivo.

[c] 2013 Elsevier GmbH. All rights reserved.

Introduction

Influenza viruses frequently cause high morbidity and significant mortality worldwide, and the lack of effective prevention and treatment stresses the importance of research for novel anti-influenza agents. Traditional medical plants have been used to develop resistance against viruses throughout history and are increasingly drawing attention as potential sources for development of antiviral drugs in recent years.

Commelina communis L., also known as dayflower, is widely distributed in the world. C communis L. has been traditionally used to treat noninfectious fever, edema, hordeolum, and diabetes in China (Youn et al. 2004), and reported to contain a number of chemical constituents, such as flavonoids, alkaloids, polysaccharides, terpenes and sterols (Yang et al. 2007).

Our preliminary in vitro study showed one of alkaloids, homonojirimycin (HNJ, the chemical structure of which is shown in Fig. 1) from C. communis L. to be effective in suppressing influenza A virus replication in Madin-Darby canine kidney (MDCK) cells (Zhang et al. 2013). The aim of this study was to investigate the effects of HNJ on protection against influenza virus infection in mice, and to evaluate its immunomodulatory potential in relation to the alleviation of influenza virus infection.

Materials and methods

Preparation of HNJ

The whole plant of C. communis L. was collected from Tortoise Hill (Wuhan, China), and was identified by Prof. Ke-Li Chen, Hubei University of Chinese Medicine. The dried herbs (3 kg) were extracted as described in our previous reports (Bing et al., 2009). Further separation was achieved through preparative HPLC (column: Asahipak NH2P, 4.6 mm i.d. x 250 mm; eluent: 80% acetonitrile; flow rate: 1.5 ml/min; UV detector: 200 nm), producing compound 1 (550 mg), which was elucidated as HNJ by spectroscopic analyses and comparison with those published previously (Bae et al. 1992; Kim et al. 1999). The purity of HNJ was greater than 95% based on gas chromatographic analysis.

Virus and reagents

MDCK cells were maintained in Eagle's minimum essential medium containing 10% calf serum and penicillin-streptomycin. Influenza A virus (A/PR/8/34 H1N1, PR8), provided by Institute of Virolog, Wuhan University, was propagated in MDCK cells. Ribavirin, a positive control, was obtained from O2 Pharmaceuticals (Zhejiang, China). Ribavirin and HNJ were dissolved in saline and further diluted to the appropriate concentration before use of experiments.

Mice

Female BALB/c mice weighing 18-22g were obtained from Hubei Laboratory Animal Research Center (Wuhan, China) and housed in a 12-h light/dark cycle under room temperature (maintained at 22 [+ or -] 2[degrees]C) and humidity range of 35-65%. The median infectious dose ([ID.sub.50]) of PR8 in mice was calculated by the method of Reed and Muench (1938). The Animal Care and Use Committee of He'nan University of Traditional Chinese Medicine approved all procedures involving animals.

Protective efficacy in mice

Mice were infected intranasally with 5 x [ID.sub.50] PR8 suspension in sterile phosphate-buffered saline (PBS) under ether anesthesia. HNJ graded doses (0.5, 1, and 2 mg/kg) or ribavirin (70 mg/kg) was orally administered to the mice two times daily from 2 days before infection to 6 days post-infection (p.i.), while the mice in the virus control group were only given saline in the same manner. In the survival time study (n = 12), the mice were monitored daily for signs of toxicity and death for 21 days p.i. The protection was estimated by reduction of mortality and prolongation of mean survival days (MSD). In the cytokine detection study (n = 18), six mice from each group were sacrificed and samples were taken on the days 2, 4 and 6 p.i. At sacrifice, blood was immediately collected and centrifuged at 3000 rpm for 15 min to obtain serum. The lungs of each mouse were harvested and homogenized in PBS, then supernatants were stored at -80[degrees]C until analysis.

Lung viral titers analysis

Total RNA was extracted from the lung homogenate using the Trizol Reagent (1 ml of Trizol for 30 mg tissue), following the manufacturer's instruction (Invitrogen, Carlsbad, CA). Real-time polymerase chain reaction (RT-PCR) reaction was carried out in a final volume of 25[micro]l, using 2.5[micro]l cDNA template, 12.5[micro]l SYBR Green Master Mix (Toyobo, Japan), 2.5[micro]l plus solution and 5.5[micro]l DEPC water, and 2[micro]l for forward and reverse primers (10[micro]M). The PCR procedure started with an initial step at 50[degrees]C for 2min then 95[degrees]C for 2min, followed by 40 cycles of denaturation at 94[degrees]C for 15s, annealing at 58-62[degrees]C for 15s and extension at 72[degrees]C for 15s. Melt curve analyses were performed at the end of each PCR. Gene expression was normalized to RNA loading for each sample using the 13-actin RNA as an internal standard. The quantity of mRNA was evaluated using a comparative [C.sub.t] method (Fink et al. 1998). Data were expressed as [10.sup.5] copies of viral genome per ml. Triplicate measurements were made for each sample. The primers for PR8 nucleoprotein gene were: forward 5'-AGTCACAATAGGAGAGTGCCCA-3' and reverse 5'-ATTCCAGTCCATCCCCCITC-3'. The primers for [beta]-actin were: forward 5'-ACCATTGGCAATGAGCGGT-3' and reverse 5'-GTCTTTGCGGATGTCCACGT-3'.

Determination of cytokines

Interferon (IFN)-[gamma], tumor necrosis factor (TNF)-[alpha], interleukin (IL)-6 and IL-10 levels in serum or lung homogenates were assessed using the enzyme-linked immunosorbent assay (ELISA) reagent kits (Boster Biotech. Inc., Wuhan, China) according to the manufacture's instructions. Briefly, each sample was incubated with the monoclonal antibody in a 96-well plate at 37[degrees]C for 2h, and then washed three times with PBS. Polyclonal antibody was added again and incubated for 2h. After three washings, tetramethylbenzidine substrate solution was added and incubated for 30 min. The absorbance was measured at 450 nm with a microplate reader.

Statistical analysis

The data were analyzed as follows: percent survival by [X.sup.2] test and MSD, virus titers and amount of cytokines by Student's t-test. Differences were considered statistically significant when p < 0.05.

Results

Effect of HNJ on the survival of influenza-infected mice

The effect of oral treatment of HNJ on the survival rate of influenza-infected mice was evaluated (Fig. 2). 83.3% of mice in the virus control group did not survive beyond 15 days p.i. On the other hand, significant improvement in the mortality rate in mice treated with HNJ or ribavirin was observed. While the MSD of virus control mice was (9.7 [+ or -] 3.2) clays, the mice administered HNJ at 1 and 2 mg/kg survived for (12.3 [+ or -] 1.5) days (p < 0.05) and (11.7 [+ or -] 0.9) days, respectively.

Effect of HNJ on the PR8 viral loads in lungs of influenza-infected mice

In the survival time experiment, a dose of HNJ 1 mg/kg showed almost the same effect as that seen at 2 mg/kg. Therefore, a minimal effective dose (1 mg/kg) was used in the lung viral titer experiment. The effect of HNJ on viral loads in lungs was examined on days 2, 4 and 6 p.i. (Fig. 3). In mice treated with saline, virus yields increased and reached a peak (76.1 x [10.sup.5] copies/ml) on day 4 and remained at a high level (60.4 x [10.sup.5] copies/ml) on clay 6 p.i. As compared to the virus control group, treatment with HNJ (1 mg/kg) significantly reduced virus yields on days 4 and 6 p.i. (p < 0.05 or p <0.01), i.e. 56.3 and 49.2 x [10.sup.5] copies/ml, respectively. Ribavirin showed significantly strong inhibition on viral titers on all days.

Effect of HNJ on the cytokines levels in serum and lungs of influenza-infected mice

Levels of antiviral cytokines, such as IFN-[gamma] and IL-10, and pro-inflammatory cytokines, such as TNF-[alpha] and IL-6 in serum and lung tissue were examined on days 2, 4 and 6 p.i. As shown in Fig. 4A, oral administration of HNJ significantly increased the production of IFN-[gamma] but reduced the level of IL-6 in serum on days 4 and 6 p.i., compared with the virus control group (p < 0.05 or p < 0.01). In addition, the concentration of serum IL-10 was markedly elevated, whereas serum TNF-[alpha] was dramatically decreased in mice treated with HNJ on days 2, 4and 6 p.i., compared with those of virus control mice (p < 0.05 or p < 0.01). Levels of IFN-[gamma], TNF-[alpha], IL-6 and IL-10 exhibited the similar tendencies as for the lung tissue for all of the groups (Fig. 4B).

Discussion

It was reported that the dayflower, C. communis L., contains a number of alkaloids, such as harman, HNJ, 1-carbomethoxy-[beta]-carboline and 2,5-dihydroxymethy1-3,4-dihydroxypyrrolidine (Bae et al. 1992; Kim et al. 1999). Recently, alkaloids have been paid attention due to their various antiviral activities against influenza virus (Chiou et al. 2011; He et al. 2012; Wu et al. 2011b; Zeng et al. 2006). We have also demonstrated that HNJ, derived from dayflower, could inhibit influenza A virus growth in vitro (Zhang et al. 2013). In the present study, oral administration of HNJ could enhance protection against PR8 infection in mice. We observed an improved survival rate and a prolonged mean survival time in mice treated with HNJ. It is well known that the reduction in virus titer is most important for reducing mortality clue to influenza virus infection (Izumo et al. 2010). HNJ administration was also effective in decreasing viral load in lungs on days 4 and 6 p.i., suggesting that virus-elimination was promoted by HNJ administration. In addition, the data showed that the 1 mg/kg of HNJ was the most effective dose for improving the survival rate, which indicate that HNJ has a suitable close range for exhibiting the therapeutic effect for this murine model of PR8 infection.

Cytokines, such as IFN-[gamma] as a Th1 cytokine, IL-10 as a Th2 cytokine and TNF-[alpha] and IL-6 as pro-inflammatory cytokines, have been shown to be produced in response to influenza viral infection models in animals (Hennet et al. 1992; Takecla et al. 2011; Van Reeth 2000). These cytokines may modify the course of infection, and play an important role in influenza virus-induced pathogenesis.

The immunoregulatory cytokine IFN-[gamma] is particularly pivotal in limiting the spread of influenza viral infection (Garcia-Sastre et al. 1998). Previous research revealed that sequential administration of IFN-[gamma] protected influenza-infected mice from death at the early stages of infection (Weiss et al. 2010). It has also been reported that the peak level of IFN-[gamma] was detected in serum and bronchoalveolar lavage fluid of influenza-infected mice on day 6 p.i. (Kurokawa et al. 2002). In addition, IL-10 is recognized as an anti-inflammatory cytokine that can regulate immune responses. Prevention of IL-10 in vivo resulted in enhanced pulmonary inflammation and lethal injury during influenza infection (Jie et al. 2009). In our study, the data showed that the expression level of IFN-[gamma] was markedly augmented on clays 4 and 6 p.i. and IL-10 on days 2, 4 and 6 p.i. by HNJ administration (1 mg/kg) in serum of infected mice. The results indicate that HNJ may prevent inflammatory responses and strengthen host resistance against viral infection by activating secretion of IFN-[gamma] and IL-10.

Pro-inflammatory cytokines. such as TNF-[alpha] and IL-6, are considered to play an essential role in the acute inflammatory responses of infection and in tissue repair. Highly increased levels of TNF-[alpha] IL-6 following viral infection are capable of causing pneumonia, viremia and anorexia, and this action can induce lethal damage (Conn et al. 1995; Kaiser et al. 2001). It has been suggested that inhibition of TNF-[alpha] could reduce the severity of virus-specific lung immunopathology (Hussell et al. 2001), and the level of 1L-6 could be used as a predictive value in the prognosis for influenza (Ichiyama et al. 2003). Wu et al. (2011a) reported that the levels of TNF-[alpha] and IL-6 in influenza-infected mice were significantly increased after infection. In our experiment, oral administration of HNJ (1 mg/kg) significantly decreased TNF-[alpha] level on days 2, 4 and 6 p.i. and IL-6 level on days 4 and 6 p.i. in serum of PR8 infected mice. These results implied that HNJ was capable of suppressing acute lung injuries and inflammation by inhibiting TNF-[alpha] and IL-6 expression, which may contribute to the increased survival rate of PR8 infected mice.

In addition, the regulating effects of HNJ administration (1 mg/kg) on lung cytokines levels were similar to those seen on serum, which may lead to the reduction of lung inflammation in vivo. Therefore, the protective effect of HNJ on lethal infection of influenza virus might be due at least in part to the up-regulated expression of IFN-[gamma] and IL-10 and the down-regulated expression of TNF-[alpha] and IL-6 levels. Similar observations on Hypericum perforatum L. extract were recently reported by Pu et al. (2012).

To conclude, our results demonstrated that HNJ, one of alkaloids from C. communis L., exerting protection against PR8 infection and producing effective immune responses in vivo, may be useful for prevention or treatment of influenza virus infection.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81102870) and Foundation of He'nan Educational Committee (12B360014).

Abbreviations: C. communis L., Commelina communis L; HNJ, homonojirimycin; IL., interleukin: INF, interferon; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; PR8. A/PR/8/34; TNF, tumor necrosis factor.

* Corresponding author. Tel.: +86 27 68890137.

E-mail address: bingfeihong@l26.com (F.-h. Bing).

0944-7113/$--see front matter [C] 2013 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.phymed.2013.04.009

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Guo-bin Zhang, (a) Lian-qi Tian, (a) Yi-ming Li, (b) Yi-fan Liao (b), Jing Li, (b) Fei-hong Bing. (b) *

(a) Pharmacy College, Henan University of Traditional Chinese Medicine, Zhengzhou 450008, China

(b) College of Pharmacy, Hubei University of Chinese Medicine, Wuhan 430065, China
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Author:Zhang, Guo-bin; Tian, Lian-qi; Li, Yi-ming; Liao, Yi-fan; Li, Jing; Bing, Fei-hong
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Aug 15, 2013
Words:3321
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