Kai Hao

Algicidal activity of Salvia miltiorrhiza Bung on Microcystis aeruginosa—Towards identification of algicidal substance and determination of inhibition mechanism

The present study was to isolate and identify a potent algicidal compound from extract of Salvia miltiorrhiza and study the potential inhibition mechanism on Microcystis aeruginosa. Column chromatography and bioassay-guided fractionation methods were carried out to yield neo-przewaquinone A, which was identified by spectral analysis. The EC50 of neo-przewaquinone A on M. aeruginosa were 4.68 mg L(-1). In addition, neo-przewaquinone A showed relatively higher security on Chlorella pyrenoidosa and Scenedesmus obliquus, with the EC50 values of 14.78 and 10.37 mg L(-1), respectively. For the potential inhibition mechanisms, neo-przewaquinone A caused M. aeruginosa cells morphologic damage or lysis, increased malondialdehyde content and decreased the soluble protein content, total antioxidant and superoxide dismutase activity, and significantly inhibited three photosynthesis-related genes (psaB, psbD, and rbcL). The results demonstrated the algicidal effect of neo-przewaquinone A on M. aeruginosa and provided the possible inhibition mechanisms.

Magnolol and honokiol from Magnolia officinalis enhanced antiviral immune responses against grass carp reovirus in Ctenopharyngodon idella kidney cells

Abstract Medicinal plants have been widely used for a long history. Exploration of pharmacologically active compounds from medicinal plants present a broad prevalent of application. By examining viral mRNA expression in GCRV‐infected Ctenopharyngodon idella kidney (CIK) cells treated with thirty kinds of plant extracts, we identified Magnolia officinalis Rehd et Wils. was able to preferably suppress viral replication. Further studies demonstrated that the main ingredients of magnolia bark, namely, magnolol and honokiol presented protective pharmacological function when treated GCRV‐infected CIK cells with a concentration of 2.00 &mgr;g/ml and 1.25 &mgr;g/ml, respectively. Furthermore, reverse transcript quantitative polymerase chain reaction (RT‐qPCR) and western blot showed that both magnolol and honokiol were efficient to restrain the replication of GCRV in CIK cells at non‐toxic concentration (2.51 ± 0.51 &mgr;g/ml for magnolol, and 3.18 ± 0.61 &mgr;g/ml for honokiol). Moreover, it was found that magnolol and honokiol promoted the expression of immune‐related genes. Magnolol obviously significantly increased the expression of interferon (IFN) regulatory factor (IRF)7 rather than that of IRF3 in the GCRV‐infected cells, leading to the activation of type I IFN (IFN‐I). Simultaneously, magnolol drastically facilitated the expression of interleukin (IL)‐1&bgr;, but failed to induce the molecules in nuclear factor (NF)‐&kgr;B pathways. Differently, honokiol strikingly motivated not only the expression of IL‐1&bgr;, but also those of tumor necrosis factor &agr; (TNF&agr;) and NF‐&kgr;B. Interestingly, though honokiol motivated the expression of IFN‐&bgr; promoter stimulator 1 (IPS‐1), IRF3 and IRF7, it failed to up‐regulate the expression of IFN‐I, indicating that honokiol enhanced the host innate antiviral response to GCRV infection via NF‐&kgr;B pathways. Collectively, the present study revealed that magnolol and honokiol facilitated the expression of innate immune‐related genes to strengthen the innate immune signaling responses to resist GCRV infection, which contributed to understanding the mechanisms by which small‐molecule drugs possessed antiviral activities. In addition, these results lay a foundation for the development of broad‐spectrum antiviral compounds in aquaculture industry. HighlightsMagnolol and honokiol showed strong antiviral effect against GCRV infection.Magnolol provoked IRF7 transcripts, leading the activation of IFN‐I.Honokiol strikingly motivated IL‐1&bgr;, TNF&agr; and NF‐&kgr;B, reinforcing the host antiviral response via NF‐&kgr;B pathways.

In vitro immunocompetence of two compounds isolated from Polygala tenuifolia and development of resistance against grass carp reovirus (GCRV) and Dactylogyrus intermedius in respective host

The present study was undertaken to isolate some compounds from methanol extract of Polygala tenuifolia and evaluate their immunostimulatory properties and antiviral activity using grass carp Ctenopharyngodon idella kidney (CIK) cells and GCRV. By applying insecticidal bioassay-guided, chromatography techniques and successive recrystallization, two purified compounds were obtained. The changes of expression of selected immune genes (Mx1, IL-1β, TNFα, MyD88 and IgM) in C. idella kidney cell lines were evaluated after exposure to these isolated compounds. The results showed that compound 1 and 2 up-regulated to varying degrees of Mx1, IL-1β, TNFα, and MyD88 in C. idella kidney cells. WST-8 kit assay verified the two compounds has no toxic effects on CIK cell, and furthermore, have in vitro antivirus activity. Especially, that there is keeping 79% cell viability when exposure to compound 2 (100 mg L(-1)). According to in vivo insecticidal assays against Dactylogyrus intermedius, compound 2 exhibited higher efficacy than compound 1, which was found to be 87.2% effective at the concentrations of 5 mg L(-1) and safe to goldfish (Carassius auratus). Besides, the purified compounds were identified by spectral data as: (1) 1,5-Anhydro-D-glucitol and (2) 3,4,5-trimethoxy cinnamic acid. Overall, the results indicate that bath administration of these compounds modulates the immune related genes in C. idella kidney cells and to some extent, eliminate the virus and parasitic infections.

Protective immunity of grass carp induced by DNA vaccine encoding capsid protein gene (vp7) of grass carp reovirus using bacterial ghost as delivery vehicles

ABSTRACT Grass carp reovirus (GCRV) is one of the most pathogenic aquareovirus and can cause lethal hemorrhagic disease in grass carp (Ctenopharyngodon idella). However, management of GCRV infection remains a challenge. Therefore, it is necessary to find effective means for the control of its infection. The uses of bacterial ghost (BG, non‐living bacteria) as carriers for DNA delivery have received considerable attentions in veterinary and human vaccines studies. Nevertheless, there is still no report about intramuscular administration of bacterial ghost‐based DNA vaccines in fish. In the current study, a novel vaccine based on Escherichia coli DH5&agr; bacterial ghost (DH5&agr;‐BG), delivering a major capsid protein gene (vp7) of grass carp reovirus encoded DNA vaccine was developed to enhance the efficacy of a vp7 DNA vaccine against GCRV in grass carp. The grass carp was injected intramuscularly by different treatments ‐i) naked pcDNA‐vp7 (containing plasmid 1, 2.5 and 5 &mgr;g, respectively), ii) DH5&agr;‐BG/pcDNA‐vp7 (containing plasmid 1, 2.5 and 5 &mgr;g, respectively) and iii) naked pcDNA, DH5&agr;‐BG or phosphate buffered saline. The immune responses and disease resistance of grass carp were assessed in different groups, and results indicated that the antibody levels, serum total antioxidant capacity (T‐AOC), superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity and immune‐related genes were significantly enhanced in fish immunized with DH5&agr;‐BG/pcDNA‐vp7 vaccine (DNA dose ranged from 2.5 to 5 &mgr;g). In addition, the relative percentage survival were significantly enhanced in fish immunized with DH5&agr;‐BG/pcDNA‐vp7 vaccine and the relative percentage survival reached to 90% in DH5&agr;‐BG/pcDNA‐vp7 group than that of naked pcDNA‐vp7 (42.22%) at the highest DNA dose (5 &mgr;g) after 14 days of post infection. Moreover, the level of pcDNA‐vp7 plasmid was higher in DH5&agr;‐BG/pcDNA‐vp7 groups than naked pcDNA‐vp7 groups in muscle and kidneys tissues after 21 days. Overall, those results suggested that DH5&agr; bacterial ghost based DNA vaccine might be used as a promising vaccine for aquatic animals to fight against GCRV infection. HIGHLIGHTSE. coli DH5&agr; ghost was used as carrier to prepare a novel DNA vaccine in grass carp.Immunization with DH5&agr;/pcDNAvp7 induced stronger immune response than naked pcDNAvp7.Immunization with DH5&agr;/pcDNAvp7 enhanced disease resistance against GCRV in fish.Bacterial ghost based DNA vaccine had prospects for application to aquatic vaccine.

Display of GCRV vp7 protein on the surface of Escherichia coli and its immunoprotective effects in grass carp (Ctenopharyngodon idella)

ABSTRACT Infection with Grass carp reovirus (GCRV) is becoming unprecedentedly widespread in grass carp (Ctenopharyngodon idella) aquaculture industry, yet the management of GCRV infection still remains a challenge. Therefore, it is of importance to develop effective means against GCRV. As a delivery system of viral antigens, surface displaying of heterologous proteins on bacteria using anchoring motifs has successfully been implemented in human and veterinary vaccines research. In this study, a novel vaccine (BL21/InpN/vp7) was developed based on surface displaying a major capsid protein (vp7) of GCRV using the anchoring motif of N‐terminal unique domain of ice‐nucleation protein (InpN) on Escherichia coli BL21 (DE3) vaccine. Then the grass carp were immunized by surface displaying BL21/InpN/vp7 vaccine against GCRV using both intraperitoneal injection and bath immunization and their immune responses were tested. The results revealed that some non‐specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T‐AOC)) were strongly increased in grass carp post injection inoculation (vp7 dose ranged from 10 to 20 &mgr;g). The specific antibody levels against GCRV and the transcriptional of immune‐related genes (TNF‐&agr;, IL‐1&bgr;, MHCI and IgM) were also significantly enhanced in grass carp by injection inoculation (vp7 dose ranged from 5 to 20 &mgr;g). On the other hand, only the highest dose of bath vaccination significantly induced the production of specific antibody and up‐regulated transcriptions of several immune‐related genes (IgM and MHCI) in grass carp. The lower cumulative mortality of grass carp in vaccinated groups after GCRV challenge clearly demonstrated that surface displayed vp7 vaccine could protect fish against GCRV infection. The relative percentage survival (RPS) value in injection vaccinated group (88.89%) was much higher compared to bath group (18.89%), which was in consistent with the production of specific serum antibodies, non‐specific immune response and immune related genes expression. To sum up, our results indicated the surface display of heterologous antigenic proteins on E. coli BL21 (DE3) using the anchoring motif of ice‐nucleation protein may provide a promising approach to the vaccine development of aquatic animals and suggested its potential to be used as vaccine to fight against GCRV infection. HighlightsGCRV vp7 protein was displayed on surface of E. coli BL21 using InpN anchor motif.Immunization by injection with BL21/InpN/vp7 strongly induced immune response in fish.Immunization with BL21/InpN/vp7 enhanced disease resistance against GCRV.Subunit vaccine based on surface display has prospect for application against GCRV.

Effects of moroxydine hydrochloride and ribavirin on the cellular growth and immune responses by inhibition of GCRV proliferation

Moroxydine hydrochloride (Mor) and ribavirin (Rib) are known for their multi-antiviral activities against DNA and RNA viruses but little information is available about the pharmacological impact in aquaculture. The present study was undertaken to investigate the response of host cells to antiviral compounds during the anti-GCRV treatment. The scanning electron microscope results showed that Ctenopharyngodon idella kidney (CIK) cells have a higher death rate at 72h post virus infection. At the concentration of 40μgmL-1, Mor and Rib had a significant protective effect on virus-infected cells. Moreover, the gene expressions of vp5, vp6 and NS66 were significantly inhibited by treatment with Mor or Rib, especially gene expression of the vp5. For the immunoregulatory action, no distinct induction of the expression of immune genes was observed after the addition of Mor and Rib to the virus-free cells. However, the compounds significantly decreased the virus-induced gene overexpression of Myd88, Mx1, IL-1β, IL-8, I-IFN and TNFα in CIK cells. Moreover, Mor and Rib significantly inhibited the immune genes upregulation which was induced by GCRV in kidney, liver, muscle and gill of grass carp, despite greater partial gene expressions were detected than the virus-free control group. Besides, Mor and Rib blocked cell cycle changes, cytopathic effects, cellular death and virus proliferation in CIK cells thereby maintaining normal morphological structure. Overall, Mor and Rib as antiviral compounds are effective for the control of GCRV replication and the indirectly regulation of cellular immune response.

Magnolol protects Ctenopharyngodon idella kidney cells from apoptosis induced by grass carp reovirus

ABSTRACT Many natural products from medicinal plants are small molecular weight compounds with enormous structural diversity and show various biological activities. Magnolol is a biphenol compound rich in the stem bark of Magnolia officinalis Rehd et Wils., and is able to suppress viral replication in GCRV‐infected grass carp (Ctenopharyngodon idella) kidney (CIK) cells in the previous study. In this study, in vivo studies demonstrated that magnolol was efficient to restrain the replication of GCRV and repair the low level of superoxide dismutase and total antioxidant capacity in serum at the non‐toxic concentration in vivo. Furthermore, magnolol inhibited CIK cell apoptosis induced by GCRV and kept the normal cellular morphological structure, reflecting in the protection of CIK cells from cell swelling, the formation of apoptotic bodies, the disappearance of cellular morphology and nuclear fragmentation. Reverse transcript quantitative polymerase chain reaction (RT‐qPCR) showed that magnolol facilitated the expression of apoptosis‐inhibiting gene bcl‐2, while suppressed the expression of apoptosis‐promoting gene bax in GCRV‐infected cells. Besides, RT‐qPCR and enzyme activity assays proved that magnolol suppressed the expression of caspase 3, caspase 8 and caspase 9. Moreover, interactions between magnolol and proteins were predicted by using the STITCH program, which revealed that ten proteins including caspase 3, were involved in the apoptosis pathway, p53 signaling pathway, mitogen‐activated protein kinase (MAPK) signaling pathway and toll‐like receptor signaling pathway. Further assays were performed to test the effect of magnolol on apoptosis pathway, which showed that magnolol dramatically inhibited the activity of caspase 3 rather than those of caspase 8 and caspase 9. Collectively, the present study revealed that magnolol heightened the resistance of grass carp against GCRV infection and refrained GCRV‐induced apoptosis, which may be attributed to the direct interaction of magnolol with caspase 3. The present results make a contribution to understanding the mechanisms by which small‐molecule drugs possess antiviral activities, and lay a foundation for the development of broad‐spectrum antiviral compounds in aquaculture industry. HIGHLIGHTSMagnolol suppressed GCRV replication and enhanced antioxidant capacity in vivo.Magnolol inhibited GCRV‐induced apoptosis and kept the normal cellular morphological structure.The potential interaction of magnolol with caspase 3 hampered GCRV‐induced apoptosis.