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Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus
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Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus Yin-guang Caoa,b , Yu Haoa , Zhi-hui Lia,b,** , Shun-tao Liua , Le-xin Wangc,* a b c
Clinical Laboratory, Liaocheng People’s Hospital of Taishan Medical University, Shandong 252000, China Shandong University School of Medicine, Shandong 250012, China School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
A R T I C L E I N F O
Article history: Received 21 August 2016 Received in revised form 19 October 2016 Accepted 27 October 2016 Keywords: Laminaria japonica Respiratory syncytial virus Polysaccharide extract Interferon-alpha Antiviral activity
A B S T R A C T
This study was designed to investigate the inhibition activity of polysaccharide extract from Laminaria japonica against RSV. The polysaccharide from Laminaria japonica was isolated by ethanol precipitation. HEK293 cells were infected with RVS, and the antiviral activity of polysaccharide extract against RSV in host cells was tested. By using ELISA and western blot assay, the expression level of IFN-a and IRF3 and their functional roles in polysaccharide-mediated antiviral activity against RSV were investigated. The polysaccharide extract from Laminaria japonica had low toxicity to HEK293 cell. The TC50 to HEK293 cells was up to 1.76 mg/mL. Furthermore, the EC50 of polysaccharide extract to RSV was 5.27 mg/mL, and TI was 334. The polysaccharide extract improved IRF-3 expression which promoted the level of IFN-a. In conclusion: Polysaccharide extract from Laminaria japonica elicits antiviral activity against RSV by upregulation of IRF3 signaling-mediated IFN-a production. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Kelp, is also named “river cabbage” and “kombu”, belong to phylum Phaeophyta, a class of Phaeospophyceae and Laminariales, which is a family of Laminariaceae. Kelp is brown in color and can grow up to 4 m in length. It is also known as “longevity food”, “Sea of vegetables”, or “iodine champion”. Kelp is farmed mostly in southeast China. It contains a high concentration of nutrients and has potential medicinal value except for its application as food and industrial raw material [1]. Kelp’s medicinal values were recorded in some ancient Chinese medicinal handbooks, such as “Jiayou Materia Medica”, where it was used to facilitate childbirth or labor, treating gynecological and endocrinology disease or cancer [2,3]. Kelp was also described to be able to inhibit viral infection and treating thyroid carcinoma [2,3]. A patent about antiviral activity of seaweed polysaccharide against Human Immunodeficiency Virus (HIV) was applied in 1988
* Corresponding author at: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia. ** Corresponding author at: Clinical Laboratory, Liaocheng People’s Hospital of Taishan Medical University, Shandong, 252000, China. E-mail addresses: [email protected] (Z.-h. Li), [email protected] (L.-x. Wang).
[4], and more studies about seaweed were reported later. TrejoAvila [5] reported that fucoidan has antiviral activity against Newcastle disease virus (NDV). Makarenkova [6] found that brown algae Laminaria japonica can inhibit the replication of H5N1 virus. Additionally, it was reported that a combination of antiviral therapy and wet external application of kelp can efficiently relieve localized pain in acute swelling in patients with mumps infection, reduce skin temperature at parotid gland, and reduce the hospital stays and improve the comfort and satisfaction of patients. In 2013, Hayashi [7] found that fucoidan from sporophyll of Undaria pinnatifida has anti-influenza A virus characteristics. Respiratory syncytial virus (RSV) consists of 10 genes encoding 11 proteins and is transmitted by direct and indirect contact with nasal or oral secretions. RSV can cause repetitive infections throughout life, and significant morbidity or mortality in pediatric and elderly populations [8,9] . Importantly, RSV is responsible for one-third of deaths resulting in the first year of life [10]. There are no effective drugs or vaccines up to date to treat or prevent RSV infection. There is little information about whether polysaccharide from Laminaria Japonica can inhibit RSV. Therefore, our study aims to isolate polysaccharide from Laminaria Japonica and investigate its antiviral activity against RSV.
http://dx.doi.org/10.1016/j.biopha.2016.10.082 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Y.- Cao, et al., Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.10.082
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1.1. Material and methods RSV and HEK293 cells were stored in our laboratory. RPI-1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen (New York, USA). Cell counting kit-8 (CCK-8) kit, protein extraction kit and nuclear protein kit were purchased from Beyotime Biotechnology (Shanghai, China). IFN-a assay kit was purchased from Dakewe Biotech (Shenzhen, China). Anti-IRF3 antibody was purchased from Abcam (Cambridge, UK). Real time RT-PCR kit and pre-stained protein marker were purchased from Thermo (Waltham, MA USA). Interference RNA and primers were synthesized Shanghai Biochemical (Shanghai, China). Other chemicals used in the study were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 1.2. Preparation of polysaccharide extract from laminaria japonica Fresh Laminaria japonica was chopped and homogenized, and was mixed with distilled water and boiled for 20 min. The solution was centrifuged at 5000 g for 10 min, trichloroacetic acid was added to the supernatant with a final concentration of 10% and allowed to standard overnight at 4 C. After incubation, the pH of the mixture was adjusted to 7.4 with 4 M NaOH, the supernatant was collected by centrifugation with 1000 g for 10 min and then identified as polysaccharide extract by Molisch reaction, the sample was finally stored at 4 C. 1.3. Virus passage and titer determination The HEK293 cell monolayers were washed once with PBS buffer and infected with 0.5 mL of RSV suspension at 37 C in 5% CO2 incubator. The cells were harvested when 80% cytopathic effect was observed under microscope, the virus within the cells was harvested by freezing and thawing three times at 20 C. After centrifugation at 10000 g for 20 min, the virus suspension was aliquoted and freezed at 80C. A ten-fold serial dilution of RSV was made with RPMI-1640 containing 2% FBS and added to HEK293 monolayers grown in 96 well plate, six replicate wells at each dilution and cells without virus infection in six replicate wells were used as a negative control. After 96 h incubation at 37 C in 5% CO2 incubator, 10 uL of CCK-8 solution was added to each well and incubated another 1 h. The absorbance at 450 nm was measured using a microplate reader and RSV 50% tissue culture infective dose (TCID50) [11] was calculated. 1.4. Cytotoxic assay of polysaccharide extract The polysaccharide extract was dissolved with dH2O and passed through a 0.45 um filter, the polysaccharide concentration was determined by anthrone-sulfuric acid method and glucose was used as a standard. The polysaccharide was serially diluted two fold with RPMI-1640 containing 2% FBS from a concentration of 3 mg/mL, and sequentially added to HEK293 cell monolayers grown in 96 well plate (100 uL/well, three replicate wells each dilution). After 96 h incubation at 37 C in 5% CO2, 10uL of CCK-8 was added to each well and incubated another 1 h, the absorbance at 450 nm was measured and the concentration leading to a 50% reduction in cell viability (TC50) was calculated according to the method reported [12]. 1.5. Inhibition assay of RSV The polysaccharide was serially diluted two fold with RPMI1640 containing 2% FBS from a concentration of 1000 mg/mL and sequentially added to cell monolayers grown in 96 well plate, 50 mL each well and three replicate wells each dilution. The virus
only or cells in 50 mL of RPMI-1640(2% FBS) was used as control, and six replicate wells each control. To perform the antiviral assay, 50 mL containing 100 TCID50 of RSV was added to experimental groups and virus control group. For cell control, 50 mL of RPMI1640 (2% FBS) was added. 96 h incubation at 37 C in 5% CO2, 10 mL of CCK-8 solution was to each well and incubated another 1 h, the absorbance at 450 nm was measured and 50% effective concentration (EC50) was calculated [13]. The therapeutic index (TI) was calculated by the ratio of TC50 to EC50. 1.6. Effect of polysaccharide extract on RSV replication The polysaccharide was used to inhibit RSV replication in HEK293 cell monolayers with a final concentration of 0, 10 mg/mL, and 500 mg/mL, respectively. 1000 TCID50 RSV was used to infect cells in each well. Virus only and cells only were used as control. After 2 h infection at 37 C in 5% CO2, the culture supernatant was discarded and the cells were washed twice with PBS, the fresh RPMI-1640(2% FBS) containing different concentration of polysaccharide (0, 10 mg/mL, 200 mg/mL) was added to corresponding wells and the cells were continuously incubated. The RNA from virus control and antiviral groups was isolated at 2 h, 8 h, 16 h, 32 h, and 64 h postinfection, RNA from cells control was isolated at 64 h postinfection. cDNA was synthesized by reverse transcription with random primers. Total viral genome copy number was determined with real-time PCR by using RSV specific primers (RSV 1: 5-TGGAAACATACGTGAACAAGC-3; RSV2: 5-ACATGGGCACCCATATTGTA-3) and cDNA as template. Meantime, pMD19-M (RSV M gene was inserted) was used as a standard, the standard curve was created by calculating copy numbers by using equation copy numbers = OD260 dilution fold 6 1014/(number of nucleotide 324) after quantifying with NanoDrop2000. The viral replication curve was created by calculating the RSV genome copy number at different time points using the standard curve [14]. 1.7. Measurement of IFN-a level by ELISA To investigate the secretion of IFN-a under the stimulation of polysaccharide extract from Laminaria japonica, HEK2934 cells were treated with different concentration of polysaccharide and the IFN-a in culture supernatant was measured by ELISA. HEK293 cells were seeded into 24-well plate (2 104/well) and incubated until the cells are confluent. The polysaccharide was serially diluted two fold with RPMI-1640 containing 2% FBS from a concentration of 1000 mg/mL and sequentially added 500 ml to cell monolayers of each well. The supernatant of each well was collected after 24 h incubation at 37 C in 5% CO2, and the IFN-a level in the supernatant was determined with enzyme linked immunosorbent assay (ELISA). 1.8. Detection of IRF3 by western blot To investigate whether polysaccharide extract from Laminaria japonica inhibits can activate the IRF3 signaling pathway, HEK293 cells were treated with different concentration of polysaccharide extract. HEK293 cells were seeded into 6-well plate and incubated until the cells are confluent. Polysaccharide extract diluted with RPMI-1640 (2% FBS) was added to 6-well plate with a final concentration of 0 mg/mL, 10 mg/mL and 500 mg/mL, respectively, and two wells for each concentration. After 24 h incubation, the whole protein and nuclear protein from cells were isolated by using protein extraction kit according manufacturer's protocol, the protein concentration was determined by using a protein quantification kit. The proteins were subjected to the SDS-PAGE and then transferred to the nitrocellulose membrane. The membrane was blocked with 5% skim milk in PBST and incubated
Please cite this article in press as: Y.- Cao, et al., Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.10.082
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Fig. 1. Molisch reaction. A: Polysaccharide extract from Laminaria japonica B: Starch control C: Solvent (H2O) control
with specific antibodies against IRF3 and GAPDH at 4 C for overnight. After overnight incubation with primary antibody, the membrane was washed three times with PBST and incubated with HRP-conjugated secondary antibody, the protein on the membrane was developed by ECL substrate. 1.9. Statistical analyses The statistical significances of group differences were determined using a Chi-square test and or student t-test with SPSS13.0 software (IBM SPSS, NY, USA). Correlations were calculated with a Spearman’s two-tailed test. p-values less than 0.05 were considered significant with a 95% confidence interval.
2. Results 2.1. Determination of cytotoxicity By using glucose as a standard, the polysaccharide concentration was determined with anthrone-sulfuric acid method and showed that there was 3.42 mg/mL polysaccharide in the extract from Laminaria japonica which is proved to be polysaccharide by the Molisch test (Fig. 1). Compare to the untreated cells, treatment of cells with polysaccharide at 3 mg/mL of concentration can induce increased particles, cell rounding, and death, indicating that polysaccharide extract is toxic to mammalian cells. Based on the CCK-8 result, it was shown that TC50 of polysaccharide extract to HEK293 cells was 1.76 mg/mL.
Fig. 2. The cytopathic effect caused by RSV. A: HEK293 cells only B: Cytopathic effect of HEK293 cells caused by RSV
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Fig. 5. The IFN-a level HEK293 cells were treated with different concentration of polysaccharide extract, the IFN-a level in culture supernatant was positively correlated with the concentration of polysaccharide extract.
2.3. Influence of polysaccharide from Laminaria japonica to virus replication As shown in Fig. 4, the growth curve of RSV from polysaccharide treatment group significantly flattens out compare to virus control group, suggested that the virus replication was decreased and high concentration of polysaccharide can significantly inhibit the virus replication. Fig. 3. The effect of polysaccharide extract on RSV-caused cytopathic effect. A. HEK293 cells only; B. RSV-caused CPE of HEK293 cells; C. The CPE of HEK293 cells caused by RSV in the presence of 250 mg/mL of polysaccharide extract.
2.2. RSV inhibition assay The serially diluted polysaccharide extract was used to treat RSV-infected cells and determine the antiviral activity of polysaccharide against RSV (TCID50: 4.5 107/mL) tested on HEK293 cells (Fig. 2). Compared to control group, treatment of infected cells with polysaccharide reduced the cytopathic effect of HEK293 cells (Fig. 3). Based on the obtained data, it was shown that EC50 was 5.27 mg/mL and TI was 334.
Fig. 4. The growth curve of RSV. Compare to the virus control (0 mg/mL of polysaccharide), growth curve of RSV flattens out when treated with polysaccharide extract (10 mg/mL and 500 mg/mL), and 500 mg/mL of polysaccharide has best inhibitory activity against RSV.
2.4. Measurement of IFN-a level Our data indicated that the IFN-a level was 39.74 pg/mL when cells were treated with 7.8 mg/mL of polysaccharide, whereas there was 144.41 pg/mL of IFN-a in supernatant when cells were treated with 1000 mg/mL of polysaccharide. As a control, there was 13.31 pg/mL of IFN-a in untreated cell culture. Polysaccharide extract from Laminaria japonica induced the secretion of IFN-a in a dose dependent manner (Fig. 5). 2.5. Detection of IRF3 by western blot Treatment of HEK293 cells with polysaccharide increased the protein level of IRF3 in cells and its nuclear translocation (Fig. 6).
Fig. 6. Detection of IRF3 by western blot. HEK293 cells were treated with different concentration (0, 10 mg/mL, 500 mg/mL) of polysaccharide extract, IRF3 protein level in whole cells and nuclear was detected by western blot, respectively. GAPDH was used as a control.
Please cite this article in press as: Y.- Cao, et al., Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.10.082
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3. Discussion RSV belongs to the family of Paramyxoviridae, subfamily of Pneumovirinae. It is a negative-sense single strand RNA virus. RSV is one of the major causes that lead to respiratory viral infection in young children and susceptible individuals, resulting in capillary bronchial pneumonia, respiratory syncytial virus pneumonia, and other lower respiratory tract infections. In addition, it also can cause allergies and asthma [15,16]. RSV can cause recurrent respiratory infections in infants, elderly adults, and immunocompromised individuals [17]. Thus, RSV infections impose huge burden to public health. Although the studies about vaccine and drug development against RSV have been reported [18,19], current treatment of RSV infection is primarily supportive care [20]. Polysaccharide from Laminaria japonica has been reported to have many biological activities, such as antitumor [21] and antithrombotic effects [22], as well as preventing oxidative stress [23,24]. Importantly, polysaccharide has antivirus activity too. Fucoidan, an extract from Laminaria japonica, was shown to have virucidal activity against influenza A/H5N1 virus and Hantavirus [3,6], and no cytotoxic activity to normal cells. The effect of Laminaria japonica on RSV has not been previously reported. In the present study, we have isolated polysaccharide component from Laminaria japonica and showed an antiviral activity of polysaccharide against RSV in vitro. Importantly, Laminaria japonica is one of the common food materials in China and has no known toxicities to human. The safety profile of Laminaria japonica was also confirmed by cytotoxic assay in our study. We have demonstrated that the TI of polysaccharide extract in this study was 334, which was significantly higher than other anti-RSV drugs [25,26]. We also demonstrated that the polysaccharide extract from Laminaria japonica can efficiently inhibit the RSV replication in a dose dependent manner, suggesting that polysaccharide extract has a great potential against RSV infection. So far, we are not aware of any other bioactive substances that have antiRSV activity higher than polysaccharide extract from Laminaria japonica. Ficipyrone A compound from Endophytic Fungi isolated from Nigerian medicinal plants is reported to show strong inhibition of the RSV, with IC50 being 4.22 1.03 mM which is close to that of ribavirin’s [27]. Small interfering RNA is an effective method in the treatment of RSV infection in vitro but further study about the safety and effect in vivo is warranted [28]. Interferon has a high antiviral activity and is one of the major host defense mechanisms against viral infections. It has been used as one of the most effective agents against viral infections [29,30]. Some antiviral agents elicit antiviral activity by enhancing the expression level of interferon [31]. Consistently, we found that polysaccharide extract from Laminaria japonica increased the secretion of IFN-a from HEK293 cells in a dose dependent manner when cells were treated with polysaccharide extract. IRF is the major factor that regulates the expression of IFN-a in signaling pathway [32]. IRF is usually expressed in cytosol and translocated to the nuclear after phosphorylation [33], thus the activation of IRF can be evaluated by determining its phosphorylation and nuclear translocation. By using Western blot, we demonstrated that treatment of HEK293 cells with polysaccharide extract enhanced the expression level of IRF3 and its translocation, suggesting that the antiviral activity of polysaccharide extract from Laminaria japonica is dependent on the upregulation of IFN-a level mediated by IRF3 signaling. In conclusion, we have successfully extracted polysaccharide from Laminaria japonica and removed protein contamination by using trichloroacetic acid. The isolated polysaccharide extract showed significant antiviral activity against RSV, and the antiviral activities were dependent on the IRF-mediated IFN-asecretion.
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Polysaccharides derived from Laminaria japonica may be used as a new agent against RSV infection. Acknowledgements This work was supported by China Postdoctoral Science Fund (No. 2014M561937) and Projects of medical and health technology development program of Shandong Province (No.2014WS0299) References [1] R.H. Kathan, Kelp extracts as antiviral substances, Ann. N. Y. Acad. Sci. 130 (1) (1965) 390–397. [2] J.E. Rosen, P. Gardiner, R.B. Saper, E.N. Pearce, K. Hammer, R.L. Gupta-Lawrence, S.L. Lee, Kelp use in patients with thyroid cancer, Endocrine 46 (1) (2014) 123– 130. [3] S.N. Pavliga, G.G. Kompanets, V.Y. Tsygankov, The experimental research (in vitro) of carrageenans and fucoidans to decrease activity of hantavirus, Food Environ. Virol. 8 (2) (2016) 120–124. [4] A. Ahmadi, S.Z. Moghadamtousi, S. Abubakar, K. Zandi, Antiviral potential of algae polysaccharides isolated from marine sources: a review, Biomed. Res. Int. 2015 (2015) 825203. [5] L.M. Trejo-Avila, R. 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Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus Yin-guang Caoa,b , Yu Haoa , Zhi-hui Lia,b,** , Shun-tao Liua , Le-xin Wangc,* a b c
Clinical Laboratory, Liaocheng People’s Hospital of Taishan Medical University, Shandong 252000, China Shandong University School of Medicine, Shandong 250012, China School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia
A R T I C L E I N F O
Article history: Received 21 August 2016 Received in revised form 19 October 2016 Accepted 27 October 2016 Keywords: Laminaria japonica Respiratory syncytial virus Polysaccharide extract Interferon-alpha Antiviral activity
A B S T R A C T
This study was designed to investigate the inhibition activity of polysaccharide extract from Laminaria japonica against RSV. The polysaccharide from Laminaria japonica was isolated by ethanol precipitation. HEK293 cells were infected with RVS, and the antiviral activity of polysaccharide extract against RSV in host cells was tested. By using ELISA and western blot assay, the expression level of IFN-a and IRF3 and their functional roles in polysaccharide-mediated antiviral activity against RSV were investigated. The polysaccharide extract from Laminaria japonica had low toxicity to HEK293 cell. The TC50 to HEK293 cells was up to 1.76 mg/mL. Furthermore, the EC50 of polysaccharide extract to RSV was 5.27 mg/mL, and TI was 334. The polysaccharide extract improved IRF-3 expression which promoted the level of IFN-a. In conclusion: Polysaccharide extract from Laminaria japonica elicits antiviral activity against RSV by upregulation of IRF3 signaling-mediated IFN-a production. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Kelp, is also named “river cabbage” and “kombu”, belong to phylum Phaeophyta, a class of Phaeospophyceae and Laminariales, which is a family of Laminariaceae. Kelp is brown in color and can grow up to 4 m in length. It is also known as “longevity food”, “Sea of vegetables”, or “iodine champion”. Kelp is farmed mostly in southeast China. It contains a high concentration of nutrients and has potential medicinal value except for its application as food and industrial raw material [1]. Kelp’s medicinal values were recorded in some ancient Chinese medicinal handbooks, such as “Jiayou Materia Medica”, where it was used to facilitate childbirth or labor, treating gynecological and endocrinology disease or cancer [2,3]. Kelp was also described to be able to inhibit viral infection and treating thyroid carcinoma [2,3]. A patent about antiviral activity of seaweed polysaccharide against Human Immunodeficiency Virus (HIV) was applied in 1988
* Corresponding author at: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, 2678, Australia. ** Corresponding author at: Clinical Laboratory, Liaocheng People’s Hospital of Taishan Medical University, Shandong, 252000, China. E-mail addresses: [email protected] (Z.-h. Li), [email protected] (L.-x. Wang).
[4], and more studies about seaweed were reported later. TrejoAvila [5] reported that fucoidan has antiviral activity against Newcastle disease virus (NDV). Makarenkova [6] found that brown algae Laminaria japonica can inhibit the replication of H5N1 virus. Additionally, it was reported that a combination of antiviral therapy and wet external application of kelp can efficiently relieve localized pain in acute swelling in patients with mumps infection, reduce skin temperature at parotid gland, and reduce the hospital stays and improve the comfort and satisfaction of patients. In 2013, Hayashi [7] found that fucoidan from sporophyll of Undaria pinnatifida has anti-influenza A virus characteristics. Respiratory syncytial virus (RSV) consists of 10 genes encoding 11 proteins and is transmitted by direct and indirect contact with nasal or oral secretions. RSV can cause repetitive infections throughout life, and significant morbidity or mortality in pediatric and elderly populations [8,9] . Importantly, RSV is responsible for one-third of deaths resulting in the first year of life [10]. There are no effective drugs or vaccines up to date to treat or prevent RSV infection. There is little information about whether polysaccharide from Laminaria Japonica can inhibit RSV. Therefore, our study aims to isolate polysaccharide from Laminaria Japonica and investigate its antiviral activity against RSV.
http://dx.doi.org/10.1016/j.biopha.2016.10.082 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Y.- Cao, et al., Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.10.082
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1.1. Material and methods RSV and HEK293 cells were stored in our laboratory. RPI-1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen (New York, USA). Cell counting kit-8 (CCK-8) kit, protein extraction kit and nuclear protein kit were purchased from Beyotime Biotechnology (Shanghai, China). IFN-a assay kit was purchased from Dakewe Biotech (Shenzhen, China). Anti-IRF3 antibody was purchased from Abcam (Cambridge, UK). Real time RT-PCR kit and pre-stained protein marker were purchased from Thermo (Waltham, MA USA). Interference RNA and primers were synthesized Shanghai Biochemical (Shanghai, China). Other chemicals used in the study were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 1.2. Preparation of polysaccharide extract from laminaria japonica Fresh Laminaria japonica was chopped and homogenized, and was mixed with distilled water and boiled for 20 min. The solution was centrifuged at 5000 g for 10 min, trichloroacetic acid was added to the supernatant with a final concentration of 10% and allowed to standard overnight at 4 C. After incubation, the pH of the mixture was adjusted to 7.4 with 4 M NaOH, the supernatant was collected by centrifugation with 1000 g for 10 min and then identified as polysaccharide extract by Molisch reaction, the sample was finally stored at 4 C. 1.3. Virus passage and titer determination The HEK293 cell monolayers were washed once with PBS buffer and infected with 0.5 mL of RSV suspension at 37 C in 5% CO2 incubator. The cells were harvested when 80% cytopathic effect was observed under microscope, the virus within the cells was harvested by freezing and thawing three times at 20 C. After centrifugation at 10000 g for 20 min, the virus suspension was aliquoted and freezed at 80C. A ten-fold serial dilution of RSV was made with RPMI-1640 containing 2% FBS and added to HEK293 monolayers grown in 96 well plate, six replicate wells at each dilution and cells without virus infection in six replicate wells were used as a negative control. After 96 h incubation at 37 C in 5% CO2 incubator, 10 uL of CCK-8 solution was added to each well and incubated another 1 h. The absorbance at 450 nm was measured using a microplate reader and RSV 50% tissue culture infective dose (TCID50) [11] was calculated. 1.4. Cytotoxic assay of polysaccharide extract The polysaccharide extract was dissolved with dH2O and passed through a 0.45 um filter, the polysaccharide concentration was determined by anthrone-sulfuric acid method and glucose was used as a standard. The polysaccharide was serially diluted two fold with RPMI-1640 containing 2% FBS from a concentration of 3 mg/mL, and sequentially added to HEK293 cell monolayers grown in 96 well plate (100 uL/well, three replicate wells each dilution). After 96 h incubation at 37 C in 5% CO2, 10uL of CCK-8 was added to each well and incubated another 1 h, the absorbance at 450 nm was measured and the concentration leading to a 50% reduction in cell viability (TC50) was calculated according to the method reported [12]. 1.5. Inhibition assay of RSV The polysaccharide was serially diluted two fold with RPMI1640 containing 2% FBS from a concentration of 1000 mg/mL and sequentially added to cell monolayers grown in 96 well plate, 50 mL each well and three replicate wells each dilution. The virus
only or cells in 50 mL of RPMI-1640(2% FBS) was used as control, and six replicate wells each control. To perform the antiviral assay, 50 mL containing 100 TCID50 of RSV was added to experimental groups and virus control group. For cell control, 50 mL of RPMI1640 (2% FBS) was added. 96 h incubation at 37 C in 5% CO2, 10 mL of CCK-8 solution was to each well and incubated another 1 h, the absorbance at 450 nm was measured and 50% effective concentration (EC50) was calculated [13]. The therapeutic index (TI) was calculated by the ratio of TC50 to EC50. 1.6. Effect of polysaccharide extract on RSV replication The polysaccharide was used to inhibit RSV replication in HEK293 cell monolayers with a final concentration of 0, 10 mg/mL, and 500 mg/mL, respectively. 1000 TCID50 RSV was used to infect cells in each well. Virus only and cells only were used as control. After 2 h infection at 37 C in 5% CO2, the culture supernatant was discarded and the cells were washed twice with PBS, the fresh RPMI-1640(2% FBS) containing different concentration of polysaccharide (0, 10 mg/mL, 200 mg/mL) was added to corresponding wells and the cells were continuously incubated. The RNA from virus control and antiviral groups was isolated at 2 h, 8 h, 16 h, 32 h, and 64 h postinfection, RNA from cells control was isolated at 64 h postinfection. cDNA was synthesized by reverse transcription with random primers. Total viral genome copy number was determined with real-time PCR by using RSV specific primers (RSV 1: 5-TGGAAACATACGTGAACAAGC-3; RSV2: 5-ACATGGGCACCCATATTGTA-3) and cDNA as template. Meantime, pMD19-M (RSV M gene was inserted) was used as a standard, the standard curve was created by calculating copy numbers by using equation copy numbers = OD260 dilution fold 6 1014/(number of nucleotide 324) after quantifying with NanoDrop2000. The viral replication curve was created by calculating the RSV genome copy number at different time points using the standard curve [14]. 1.7. Measurement of IFN-a level by ELISA To investigate the secretion of IFN-a under the stimulation of polysaccharide extract from Laminaria japonica, HEK2934 cells were treated with different concentration of polysaccharide and the IFN-a in culture supernatant was measured by ELISA. HEK293 cells were seeded into 24-well plate (2 104/well) and incubated until the cells are confluent. The polysaccharide was serially diluted two fold with RPMI-1640 containing 2% FBS from a concentration of 1000 mg/mL and sequentially added 500 ml to cell monolayers of each well. The supernatant of each well was collected after 24 h incubation at 37 C in 5% CO2, and the IFN-a level in the supernatant was determined with enzyme linked immunosorbent assay (ELISA). 1.8. Detection of IRF3 by western blot To investigate whether polysaccharide extract from Laminaria japonica inhibits can activate the IRF3 signaling pathway, HEK293 cells were treated with different concentration of polysaccharide extract. HEK293 cells were seeded into 6-well plate and incubated until the cells are confluent. Polysaccharide extract diluted with RPMI-1640 (2% FBS) was added to 6-well plate with a final concentration of 0 mg/mL, 10 mg/mL and 500 mg/mL, respectively, and two wells for each concentration. After 24 h incubation, the whole protein and nuclear protein from cells were isolated by using protein extraction kit according manufacturer's protocol, the protein concentration was determined by using a protein quantification kit. The proteins were subjected to the SDS-PAGE and then transferred to the nitrocellulose membrane. The membrane was blocked with 5% skim milk in PBST and incubated
Please cite this article in press as: Y.- Cao, et al., Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.10.082
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Fig. 1. Molisch reaction. A: Polysaccharide extract from Laminaria japonica B: Starch control C: Solvent (H2O) control
with specific antibodies against IRF3 and GAPDH at 4 C for overnight. After overnight incubation with primary antibody, the membrane was washed three times with PBST and incubated with HRP-conjugated secondary antibody, the protein on the membrane was developed by ECL substrate. 1.9. Statistical analyses The statistical significances of group differences were determined using a Chi-square test and or student t-test with SPSS13.0 software (IBM SPSS, NY, USA). Correlations were calculated with a Spearman’s two-tailed test. p-values less than 0.05 were considered significant with a 95% confidence interval.
2. Results 2.1. Determination of cytotoxicity By using glucose as a standard, the polysaccharide concentration was determined with anthrone-sulfuric acid method and showed that there was 3.42 mg/mL polysaccharide in the extract from Laminaria japonica which is proved to be polysaccharide by the Molisch test (Fig. 1). Compare to the untreated cells, treatment of cells with polysaccharide at 3 mg/mL of concentration can induce increased particles, cell rounding, and death, indicating that polysaccharide extract is toxic to mammalian cells. Based on the CCK-8 result, it was shown that TC50 of polysaccharide extract to HEK293 cells was 1.76 mg/mL.
Fig. 2. The cytopathic effect caused by RSV. A: HEK293 cells only B: Cytopathic effect of HEK293 cells caused by RSV
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Fig. 5. The IFN-a level HEK293 cells were treated with different concentration of polysaccharide extract, the IFN-a level in culture supernatant was positively correlated with the concentration of polysaccharide extract.
2.3. Influence of polysaccharide from Laminaria japonica to virus replication As shown in Fig. 4, the growth curve of RSV from polysaccharide treatment group significantly flattens out compare to virus control group, suggested that the virus replication was decreased and high concentration of polysaccharide can significantly inhibit the virus replication. Fig. 3. The effect of polysaccharide extract on RSV-caused cytopathic effect. A. HEK293 cells only; B. RSV-caused CPE of HEK293 cells; C. The CPE of HEK293 cells caused by RSV in the presence of 250 mg/mL of polysaccharide extract.
2.2. RSV inhibition assay The serially diluted polysaccharide extract was used to treat RSV-infected cells and determine the antiviral activity of polysaccharide against RSV (TCID50: 4.5 107/mL) tested on HEK293 cells (Fig. 2). Compared to control group, treatment of infected cells with polysaccharide reduced the cytopathic effect of HEK293 cells (Fig. 3). Based on the obtained data, it was shown that EC50 was 5.27 mg/mL and TI was 334.
Fig. 4. The growth curve of RSV. Compare to the virus control (0 mg/mL of polysaccharide), growth curve of RSV flattens out when treated with polysaccharide extract (10 mg/mL and 500 mg/mL), and 500 mg/mL of polysaccharide has best inhibitory activity against RSV.
2.4. Measurement of IFN-a level Our data indicated that the IFN-a level was 39.74 pg/mL when cells were treated with 7.8 mg/mL of polysaccharide, whereas there was 144.41 pg/mL of IFN-a in supernatant when cells were treated with 1000 mg/mL of polysaccharide. As a control, there was 13.31 pg/mL of IFN-a in untreated cell culture. Polysaccharide extract from Laminaria japonica induced the secretion of IFN-a in a dose dependent manner (Fig. 5). 2.5. Detection of IRF3 by western blot Treatment of HEK293 cells with polysaccharide increased the protein level of IRF3 in cells and its nuclear translocation (Fig. 6).
Fig. 6. Detection of IRF3 by western blot. HEK293 cells were treated with different concentration (0, 10 mg/mL, 500 mg/mL) of polysaccharide extract, IRF3 protein level in whole cells and nuclear was detected by western blot, respectively. GAPDH was used as a control.
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3. Discussion RSV belongs to the family of Paramyxoviridae, subfamily of Pneumovirinae. It is a negative-sense single strand RNA virus. RSV is one of the major causes that lead to respiratory viral infection in young children and susceptible individuals, resulting in capillary bronchial pneumonia, respiratory syncytial virus pneumonia, and other lower respiratory tract infections. In addition, it also can cause allergies and asthma [15,16]. RSV can cause recurrent respiratory infections in infants, elderly adults, and immunocompromised individuals [17]. Thus, RSV infections impose huge burden to public health. Although the studies about vaccine and drug development against RSV have been reported [18,19], current treatment of RSV infection is primarily supportive care [20]. Polysaccharide from Laminaria japonica has been reported to have many biological activities, such as antitumor [21] and antithrombotic effects [22], as well as preventing oxidative stress [23,24]. Importantly, polysaccharide has antivirus activity too. Fucoidan, an extract from Laminaria japonica, was shown to have virucidal activity against influenza A/H5N1 virus and Hantavirus [3,6], and no cytotoxic activity to normal cells. The effect of Laminaria japonica on RSV has not been previously reported. In the present study, we have isolated polysaccharide component from Laminaria japonica and showed an antiviral activity of polysaccharide against RSV in vitro. Importantly, Laminaria japonica is one of the common food materials in China and has no known toxicities to human. The safety profile of Laminaria japonica was also confirmed by cytotoxic assay in our study. We have demonstrated that the TI of polysaccharide extract in this study was 334, which was significantly higher than other anti-RSV drugs [25,26]. We also demonstrated that the polysaccharide extract from Laminaria japonica can efficiently inhibit the RSV replication in a dose dependent manner, suggesting that polysaccharide extract has a great potential against RSV infection. So far, we are not aware of any other bioactive substances that have antiRSV activity higher than polysaccharide extract from Laminaria japonica. Ficipyrone A compound from Endophytic Fungi isolated from Nigerian medicinal plants is reported to show strong inhibition of the RSV, with IC50 being 4.22 1.03 mM which is close to that of ribavirin’s [27]. Small interfering RNA is an effective method in the treatment of RSV infection in vitro but further study about the safety and effect in vivo is warranted [28]. Interferon has a high antiviral activity and is one of the major host defense mechanisms against viral infections. It has been used as one of the most effective agents against viral infections [29,30]. Some antiviral agents elicit antiviral activity by enhancing the expression level of interferon [31]. Consistently, we found that polysaccharide extract from Laminaria japonica increased the secretion of IFN-a from HEK293 cells in a dose dependent manner when cells were treated with polysaccharide extract. IRF is the major factor that regulates the expression of IFN-a in signaling pathway [32]. IRF is usually expressed in cytosol and translocated to the nuclear after phosphorylation [33], thus the activation of IRF can be evaluated by determining its phosphorylation and nuclear translocation. By using Western blot, we demonstrated that treatment of HEK293 cells with polysaccharide extract enhanced the expression level of IRF3 and its translocation, suggesting that the antiviral activity of polysaccharide extract from Laminaria japonica is dependent on the upregulation of IFN-a level mediated by IRF3 signaling. In conclusion, we have successfully extracted polysaccharide from Laminaria japonica and removed protein contamination by using trichloroacetic acid. The isolated polysaccharide extract showed significant antiviral activity against RSV, and the antiviral activities were dependent on the IRF-mediated IFN-asecretion.
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Polysaccharides derived from Laminaria japonica may be used as a new agent against RSV infection. Acknowledgements This work was supported by China Postdoctoral Science Fund (No. 2014M561937) and Projects of medical and health technology development program of Shandong Province (No.2014WS0299) References [1] R.H. Kathan, Kelp extracts as antiviral substances, Ann. N. Y. Acad. Sci. 130 (1) (1965) 390–397. [2] J.E. Rosen, P. Gardiner, R.B. Saper, E.N. Pearce, K. Hammer, R.L. Gupta-Lawrence, S.L. Lee, Kelp use in patients with thyroid cancer, Endocrine 46 (1) (2014) 123– 130. [3] S.N. Pavliga, G.G. Kompanets, V.Y. Tsygankov, The experimental research (in vitro) of carrageenans and fucoidans to decrease activity of hantavirus, Food Environ. Virol. 8 (2) (2016) 120–124. [4] A. Ahmadi, S.Z. Moghadamtousi, S. Abubakar, K. Zandi, Antiviral potential of algae polysaccharides isolated from marine sources: a review, Biomed. Res. Int. 2015 (2015) 825203. [5] L.M. Trejo-Avila, R. 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Please cite this article in press as: Y.- Cao, et al., Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.10.082