Abstract

Objective: To evaluate apoptotic activity of Viviparous bengalensis flesh extracted purified fraction VB-P4 on fibroblast like synoviocyte cells (FLS).

Methods: Purified fraction VB-P4 was analyzed for apoptotic activity on synoviocyte cell. The cells were treated with VB-P4 (40 g/mL) for 24 hrs. Morphological analysis through phase contrast microscope, MTT assay for cytotoxicity study, interleukins level analysis, CD3, CD4, CD8 activities study through flow cytometry, apoptotic activity study using annexin V- FITC, immunofluorescence study of caspase 3, and agarose gel for DNA breakage were done using synoviocyte cell.

Results: VB-P4 treatment on synovial cell showed lack of matured synoviocyte cell with respect to osteoarthritic control cells. MTT assay showed significant decrease in formazan complex formation after treatment with VB-P4 in dose dependent manner. Decreased levels of interleukins (TNF-α, IL-1α, CINC-1, IL-17, MMP-1, Cathepsin K) on synoviocyte cells also confirmed reduced osteoarthritis after treatment with VB-P4 (40 µg/mL). Flow cytometric analysis of monoclonal antibody (CD3, CD4, and CD8) exhibited increased expression on synovial cell surface after treatment with VBP4. Annexin-V FITC study showed 76.90% early apoptosis and 7.95% late apoptotic cell after treatment with VB-P4 which confirmed apoptotic activity of VB-P4 on osteoarthritic synoviocyte cells. Agarose gel electrophoresis showed that VB-P4 (40 µg/mL) worked on nuclear localization of synoviocyte which is shown by smeary bands and florescence cell micrograph. Upregulation of caspase 3 confirmed apoptosis due to VB-P4 treatment on synoviocyte cell.

Conclusion: The findings showed that fresh water snail flesh extracted purified fraction VB-P4, exhibited apoptotic activity on osteoarthritic synoviocyte cell through caspase mediated pathway.

Keywords: VB-P4, synoviocyte cells, apoptosis, flow cytometry, immunofluorescence, caspase activity.

Graphical Abstract