Abstract
Cellular internalization and intracellular trafficking of particles depend on their specific physicochemical properties. Godanti Bhasma (GB) is a traditional Indian medicine formulation prepared by heating of gypsum powder with herbal extracts. Chemically, GB is anhydrous calcium sulfate. During formulation it obtains unique physicochemical properties that lead to its rapid cellular internalization and induction of massive cytoplasmic vacuolation. Interestingly, no cellular internalization was found with parent gypsum particle. Flow cytometry analysis and live tracking of GB treated cell showed particle internalization, vacuole formation, particle dissolution and later vacuolar turnover. GB particle dissolution in acidic cell free solution mimics intravacuolar environment where GB was disintegrated under acidic pH suggesting lysosomal enzymes might have no role in GB degradation. Vacuolation often accompany with the sign of cell death whereas, in our study, massive vacuolation by GB did not induce any cell death. Moreover, GB treated cells survive with complete vacuolar process, which was reversed following post-treatment with vacuole inhibitors in GB treated cells, suggesting normal vacuolar function is essential for cell survival. In immunoblotting, upregulation of LC3-II was found in GB treated cells. Treatment of the cells with GB was also found to induce translocation of the LC3 protein from the nucleus to vacuolar membrane by immune-cytochemistry, indicating LC3 associated phagocytosis (LAP) like function. This was found to be reversed in the cells treated with vacuole inhibitors. The vacuolar function essential for cell survival, preserves mechanistic correlation with LC3 lipidation on vacuolar membrane, intracellular controlled and slow degradation of GB particle and further vacuolar turnover limiting swelling pressure in cells.
