2a).
In contrast, 17αPSCE, a synthetic progesterone derivative, had a stronger anti-H. pylori action than progesterone, and the CFUs were below the limits of detection when the organisms were cultured for 24 h with 17αPSCE at a 10 μM concentration (Fig. 2b). Incidentally, caproic acid, a constituent of 17αPSCE, did not affect the viability of H. pylori even when added to the cell suspension at a 100 μM concentration selleck compound (data not shown). Next, we measured the OD660 nm in the cell suspensions after the H. pylori (108 CFU mL−1) was incubated for 24 h with progesterone (100 μM) or 17αPSCE (100 μM) in a simple-PPLO broth (3 mL). As it turned out, the OD660 nm of the cell suspension incubated with progesterone or 17αPSCE declined to less than half of that in the control cell suspension of the H. pylori incubated in the absence of steroid http://www.selleckchem.com/products/MS-275.html (data not shown). These results suggest that H. pylori cells are lysed by the action of progesterone and 17αPSCE. Next, we carried out a series of experiments to examine whether progesterone and 17αPSCE induce the cell lysis of H. pylori via membrane injury. When PBS was used in place of the simple-PPLO broth, the CFUs of H. pylori incubated for 5 h with progesterone (100 μM) were conspicuously reduced in comparison with the baseline CFU before the incubation (Fig. 3a). The control CFUs of H. pylori incubated for 5 h without steroids were also reduced
in comparison with the baseline CFU, but the magnitude of reduction was smaller in the control CFUs than in the CFUs observed in the H. pylori incubated with progesterone. When the H. pylori was incubated for 5 h with 17αPSCE (100 μM) in PBS, the CFUs declined sharply, nearly reaching the limits of detection. The proteins in the cell supernatant these (PBS: 10 mL) obtained from the H. pylori incubated for 5 h with progesterone (100 μM) or 17αPSCE (100 μM) were analyzed by SDS-PAGE (Fig. 3b). The protein bands detected in the cell supernatant of H. pylori incubated with progesterone or 17αPSCE were considerably denser
than the protein bands detected in the control cell supernatant of H. pylori incubated without steroid. A band for flavodoxin (FldA) was found among the other protein bands. The amounts of FldA protein detected in the cell supernatant correlated closely with the decreases of CFU: the FldA protein band became more noticeable when the CFU decreased by a greater magnitude. As FldA is an electron acceptor of the oxidoreductase that catalyzes acetyl-CoA synthesis in H. pylori cell (Hughes et al., 1995), we can assume that FldA is the intracellular protein. These results, thus, suggest that progesterone and 17αPSCE exert deleterious effects on the cell membrane of H. pylori and induce cell lysis more promptly than autolysis, resulting in abundant leakage of intracellular proteins (especially FldA protein) outside of the cells.