Figure 10.

Detection of apoptosis in CECs. (A) The results of TUNEL assay showed that apoptosis of CECs was accelerated by MNU at 6 hours (P < 0.0001, n = 3), which was related to the fact that MNU promoted the expression of Bax, caspase-3, and caspase-9 and inhibited the expression of Bcl-2 (Bax: P < 0.0001, n = 3; Bcl-2: P = 0.0721, n = 3; caspase-3: P < 0.05, n = 3; and caspase-9: P < 0.01, n = 3). H₂ could reduce apoptosis by inhibiting the expression of Bax, caspase-3, and caspase-9, and promoting the expression of Bcl-2 at 6 hours (Bax: P < 0.0001, n = 3; Bcl-2: P = 0.4250, n = 3; caspase-3: P = 0.0956, n = 3; caspase-9: P < 0.01, n = 3; and TUNEL: P < 0.0001, n = 3). (B) The results of TUNEL assay showed that apoptosis of CECs was accelerated by MNU at 24 hours (P < 0.0001, n = 3), which was related to the fact that MNU promoted the expression of Bax, caspase-3, and caspase-9 and inhibited the expression of Bcl-2 (Bax: P < 0.001, n = 3; Bcl-2: P < 0.01, n = 3; caspase-3: P < 0.001, n = 3; and caspase-9: P < 0.05, n = 3). H₂ could reduce apoptosis by inhibiting the expression of Bax, caspase-3, and caspase-9 and promoting the expression of Bcl-2 at 24 hours (Bax: P < 0.05, n = 3; Bcl-2: P < 0.01, n = 3; caspase-3: P < 0.001, n = 3; caspase-9: P < 0.05, n = 3; and TUNEL: P < 0.01, n = 3). (C) WB was conducted on the related indicators of apoptosis of CECs. It was suggested that MNU could significantly increase Bax and decrease Bcl-2, with statistical significance (Bax: P < 0.001, n = 3; and Bcl-2: P < 0.01, n = 3). Apoptotic markers were detected and both caspase-3 and caspase-9 were activated by MNU (caspase-3: P < 0.001, n = 3; and caspase-9: P < 0.01, n = 3). H₂ could antagonize the pro-apoptotic effect of MNU by reducing Bax and increasing Bcl-2 (Bax: P < 0.01, n = 3; and Bcl-2: P < 0.01, n = 3), and changes of caspase-3 and caspase-9 also suggested the inhibitory effect of H₂ on the pro-apoptotic properties of MNU (caspase-3: P < 0.01, n = 3; and caspase-9: P < 0.01, n = 3).