| Purpose: To explore the inhibitory activity of Lactobacillus Salivarius ssp. salivarius JCM1231(L.Salivarius JCM1231) against Fusarium solani(F.solani) and the effects of the L. Salivarius culture filtrate(LSCF) on murine keratocytes(MKs) infected with F.solani.Methods: 1. After the growth curve was finished, L.Salivarius JCM1231 was cultured in an anaerobic incubator for 24 h, and the LSCF was prepared. Meanwhile, F.solani was cultured and spore suspension was prepared. 2. The antifungal activity of L.Salivarius JCM1231 against F.solani was determined with a plate overlay assay, agar diffusion assay and conidial germination inhibition test. 3. The effects of temperature,p H, and proteolytic enzymes on the antifungal activity of LSCF were detected with a microtiter plate well assay and conidial germination inhibition assay. 4. Build the infection model and detect the safe and effective concentration of LSCF with methylthiazoletetrazolium assays(MTT). 5. Furthermore, the effects of LSCF on MKs infected with F.solani were detected. Cell activity and apoptosis were measured using MTT assay and flow cytometry analysis, respectively. The levels of IL-6 and TNF-?cytokines were measured using quantitative real-time polymerase chain reactions(RT-q PCR) and enzyme-linked immunosorbent assays(Elisa).Results: 1. L.Salivarius JCM1231 was a kind of gram-positive bacterium, the growth of which showed “S”curve. Milk-white colony had a uniform size about 0.5~1mm in diameter; 2. According to the plate overlay assay,the mean area of the clear zones of per streak recorded was 6.225±0.895 cm2, accounting for 9.8% of the whole plate area,which suggested a strong inhibitory effect. In the agar-well diffusion assay, the mean inhibitory diameter of the LSCF group and MRS group were 22.475±0.767 mm and3.855±0.035 mm, respectively; In the conidial germination inhibition assay, the inhibition rates of conidial germination of the LSCF and MRS group were 0.995±0.007 and 0, respectively,and the mean number of germinated spores of the LSCF and MRS group were 0.5±0.7 and 96±0, respectively. Statistical difference can be detected when compare LSCF group with MRS group(P<0.05). 3. The antifungal substances produced by L.Salivarius JCM1231 were heat unstable, proteinaceous and sensitive to proteolytic enzymes and were active within a narrow acidic p H range between 2.0 and 4.0. 4. After co-incubated with 8-folds F.solani for 6h, the cell activity of MKs was decreased by36%. With MTT assays, 0 ~ 15?g/ml was determined as the safe concentration of LSCF, and 5?g/ml was determined as the min-effective concentration of LSCF.15ug/ml was accepted as the safe concentration for MKs and the effective one for the inhibition of F.solani; 5. In the presence of 15?g/ml of LSCF, cell activity was significantly increased, and cell apoptosis, the level of IL-6 and TNF-? were decreased significantly in MKs infected with F. solani. Statistical difference can be detected when compare F.solani+/LSCF+ group with F.solani+/LSCF- group(P <0.05).Conclusion: L.Salivarius JCM1231 may effectively inhibit conidial germination and mycelia growth of F.solani and the antifungal substances produced by L.Salivarius JCM1231 were heat unstable, proteinaceous and sensitive to proteolytic enzymes.What's more, LSCF can protect MKs against F.solani infection by enhancing the cell activity, inhibiting the cell apoptosis and reducing the secretion of inflammatory cytokines. This may provide new ideas for the treatment of fungal keratitis. |
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