Treatment with A. veronii supernatant led to disorganisation of actin filaments and nuclear condensation was also observed (Figure 4b2 & 4c2). However, pre-incubation of cells with VR1 supernatant maintained the Adavosertib ic50 cellular morphology comparable to control cells. In both the treatments i.e. VR1 CFS, and A. veronii CFS treatment on cells that were pre-incubated with CFS of VR1, actin filaments were present in high density at the apical perijunctional regions, encircling the
cells in a belt like manner (Figure 4b3 & 4b5). However, co-incubation of A. veronii and VR1 supernatant (Figure 4a4 to 4d4) led to the loss of membrane architecture with loss of fluorescence of ZO-1 and actin, as observed in A. veronii treatment group. Figure 4 Prevention of membrane INCB024360 supplier damage caused due to A. veronii by pre-incubated with CFS of VR1. Epithelial damage observed by immunofluorescence of tight junction proteins ZO-1 and F-actin in MDCK cell line. a) ZO-1 b) Actin c) DAPI d) Merged images for different treatment groups: 1) control, 2) A. veronii 3) VR1 4) buy IWR-1 co-incubation of VR1 with A. veronii 5) pre-incubation of VR1 with A. veronii. Pre-incubation of VR1 prevents epithelial damage due to A. veronii as observed in the merged image. Scale denotes 20 μm in all images. Figure 5 Effect
of VR1 culture supernatant in preventing the loss of cell viability caused due to A. veronii. MTT assay was performed to quantify percentage cell viability with treatment of supernatant of A. veronii and VR1, in 1:10 ratio. Cell viability graph demonstrates that the pre-incubation with VR1 supernatant
for 6 h significantly increased the cell viability. Statistical significance was determined by two tailed student’s t-test (n = 3 ± SEM, *p < 0.05). CFS of VR1 significantly lowered cytotoxicity induced by A. veronii The cytotoxic effect of A. veronii CFS was confirmed by MTT assay, which essentially checks cell viability (Figure 5). Cell viability was reduced to 60% in Vero cells treated with A. veronii supernatant for 10 h. Interestingly, Vero cells when pre-incubated with VR1 CFS for 6 h followed by 10 h of treatment with A. veronii CFS showed no loss of cell viability. SPTLC1 Similarly, VR1 CFS treatment did not show any detrimental effects on cells with no loss in cell viability. However, co-incubation of VR1 and A. veronii supernatant was not effective in preventing cytotoxicity caused by A. veronii. Discussion Kutajarista is an Ayurvedic formulation prescribed for the treatment of dysentery, piles etc. Initial characterisation of bacterial diversity of Kutajarista by the 16S rRNA gene clone library [GenBank: HQ875575-HQ875614] provided evidence about the richness of Lactobacillus spp. in the preparation of ayurvedic medicine. Therefore, the current study was aimed at characterization of probiotic and antibacterial properties of L.