Inhibition of silkworm vacuolar-type ATPase activity by its inhibitor Bafilomycin A1 induces caspase-dependent apoptosis in an embryonic cell line of silkworm

Vacuolar-type ATPase (V-ATPase) is a type of hydrogen ion transporter located in the vesicular membrane-like system, which mediates active transport and intracellular acidification in various compartments. In mammals, V-ATPase has been reported to play a key role in cell proliferation and apoptosis. The studies of V-ATPase in silkworm mainly focus on the acidification regulation of midgut and silk gland and immune resistance. However, there are few reports about the function of silkworm V-ATPase on cell proliferation, autophagy, and apoptosis. Thus, the function of V-ATPase in a cell line of Bombyx mori (BmE) was investigated by treating the cell line with bafilomycin A1, a specific inhibitor of V-ATPase. Cell counting kit 8 (CCK8) and flow cytometry analysis showed that bafilomycin A1 treatment decreased the cell proliferation activity, affected the cell cycle progression and induced cell apoptosis. LysoTracker Red staining showed that the target of bafilomycin A1 is lysosome. The expression of all autophagy-related genes (BmATG5, BmATG6, and BmATG8) decreased, indicating that cell autophagy was inhibited. The analysis of the apoptosis pathway demonstrated that inhibiting the activity of V-ATPase of BmE cells could promote mitochondria to release cytochrome C, inhibit the expression of BmIAP, and activate the caspase cascade to induce apoptosis. All these findings systematically illustrate the effects of V-ATPase on the proliferation, autophagy, and apoptosis in BmE cells, and provide new ideas and a theoretical basis for further study on the function of V-ATPase in BmE.