Mechanism of nanobacteria promoting apoptosis of human gallbladder epithelial cells

Abstract

Objective To observe the interaction between nanobacteria (NB) and human gallbladder epithelial cells, and explore the possible mechanism of porcelain gallbladder formation. Methods Absorbing bile from porcelain gallbladder and normal gallbladder to culture nanobacteria, the morphology of NB was observed under an electron microscope. Primary cultured human gallbladder epithelial cells were obtained by cutting out some normal gallbladders, and the expression of Cytokeratin-19 (CK19) was detected by immunofluorescence. NB of 1.0 Macfarland (MCF) was induced into human gallbladder epithelial cells at 0, 12, 24, 48 h. Cell damage and apoptosis rate were detected by Cell Counting Kit-8 (CCK-8), lactate dehydrogenase (LDH) kit and flow cytometry. Protein expression of B-lymphoma-2 (bcl-2), bcl-2 related X protein (bax), cytochrome C (Cyt-C), and cleaved Caspsae-9 were detected by Western blotting. Results The positive rate of NB in porcelain gallbladder and normal gallbladder bile was 88.9% and 15.0%. The difference was statistically significant (χ2=14.39, P<0.01). Under transmission electron microscopy, NB is round and unequal in size and has a diameter of about 80 nm. Immunofl-uorescence showed that CK19 protein was positive in primary cultured human gallbladder epithelial cells. NB induced human gallbladder epithelial cells at 0, 12, 24, 48 h, cell survival rate decreased significantly at 24, 48 h, the difference was statistically significant (F=305.501, P<0.01), the release of LDH began to increase significantly at 12 h, and the difference was statistically significant (F=5351.109, P<0.05). The apoptosis rate increased significantly at 24 h and 48 h, and the difference was statistically significant (F=597.457, P<0.01). The expression of bcl-2/bax was significantly decreased by Western blotting at 24 h and 48 h, and the difference was statistically significant (F=1 336.833, P<0.01). The relative expression of Cty-c was significantly increased at 12, 24, 48 h, and the difference was statistically significant (F=1 422.101, P<0.01). Cleved Caspsae-9 increased significantly at 24 h and 48 h, and the difference was statistically significant (F=2 348.061, P<0.01). Conclusion NB promotes apoptosis of human gallbladder epithelial cells through the mitochondrial pathway, which may be closely related to the formation of porcelain gallbladder. Key words: Nanobacteria; Human gallbladder epithelial cells; Apoptosis; Porcelain gallbladder