ANTIAPOPTOSIS ACTIVITY OF PLANT POLYPRENYLPHOSPHATE AGAINST MACROPHAGE TARGET CELLS INFECTED WITH THE MURINE ENCEPHALOMYELITIS VIRUS

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Introduction. Infection caused by the Theiler’s murine encephalomyelitis virus (TMEV) is regarded as an experimental model of multiple sclerosis, since both of these diseases are characterized by similar pathology of the central nervous system tissues and involvement of the immune system in the demielinization development [8]. The aim of the work was to study the effect of plant-derived polyprenylphosphate (PP) on the apoptosis of infected target cells. Material and methods. We used the drug Gamapren (LLC “Gamavetfarm”), containing 5 mg/ml of PP obtained from mulberry leaves. TMEV (strain GDVII) was titrated in BHK 21 culture. The initial titer of the virus was 106PFU/ml. P388 D1 murine macrophage-like cell line was obtained from the American Cell Culture Collection (USA). The cells were cultured in RPMI1640 medium with 10% calf embryonic serum (Gibco). After 24 hours of incubation, the cells were infected with TMEV at a dose of 20-30 PFU/cell. PP (100 µg/ml) was then introduced into half of the infected cultures. The fluorescent dye Hoechst 33342 (5 µg/ml) was added into all cultures 30 minutes before the fixation of infected cells in order to detect apoptosis-specific changes in chromatin. After 5 hours of adsorption when pronounced signs of cytopathic action were manifested (cell swelling and chromatin condensation), the cells were fixed with 4% paraformaldehyde, then permeabilized with 0.5% Triton X-100 solution. Apoptotic cells were counted visually in an inverted light microscope Leica. Results and discussion. We found that PP decreased viral-induced apoptosis of target cells. In 24 hours after TMEV infection, the number of apoptotic cells was 22%, and reached 92% 48 hours after infection. The inoculation of PP reduced the number of apoptotic cells, respectively, to 5% and 29% in 24 and 48 hours after infection. Earlier studies have shown the ability of plant-derived PP to inhibit the reproduction of DNA and RNA viruses in sensitive cell cultures [3, 4]. TMEV causes severe infection in mice, accompanied by the virus persistence in the central nervous system and the development of neuronal demielinization [8]. Reproduction of TMEV in Р338 D1 cells results in the development of apoptosis accompanied by fragmentation of the chromatin [8]. Here we showed that PP significantly reduced the number of apoptotic cells in the infected cell culture. In patients with multiple sclerosis, an increase in the level of pro-inflammatory cytokines is observed as well as a focus of inflammatory demyelination. For therapy of this disease agents with antiviral, anti-inflammatory, and antioxidant activity are used [9]. It is shown that plant-derived PPs, widely used in veterinary practice as antiviral and immunomodulatory medicines, are able to stimulate major cytokines production [2], and counteract various stress effects in vitro [7], as well as exhibit anti-inflammatory [1, 5] and antioxidant [6] effects.

About the authors

T N Kozhevnikova

The Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Moscow

A V Sanin

The Gamaleya Scientific Research Institute of Epidemiology and Microbiology, Moscow

S V Ozherelkov

M.P.Chumakov Federal Scientific Center for Research and Development of Immunobiological Preparations, Moscow region

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