ABSTRACT
[ ABSTRACT] AIM:To observe the effects of berberine and yohimbine on splenocyte apoptosis in septic mice and underlying mechanisms.METHODS:The mice were subjected to cecal ligature and puncture ( CLP) .The drugs or vehi-cle were given intragastrically 2 h after the surgery according to the following 5 groups:sham, CLP, CLP+berberine, CLP+yohimbine, and CLP+berberine+yohimbine.The apoptosis of splenocytes stained by TUNEL was observed under laser scanning confocal microscope 20 h after CLP.The splenic lymphocytes were isolated and observed using flow cytometry. The activities of caspase-3, caspase-8 and caspase-9 in splenic lymphocytes were detected, and the expression of Fas, Bim, Bcl-2 and Bax in the splenocytes was also determined by Western blotting.RESULTS:The TUNEL staining showed that the apoptotic rate of the splenocytes in septic mice 20 h after CLP was significantly higher than that in sham and CLP+yohimbine groups (P<0.05).Compared with CLP group, the proportion of apoptotic cells was decreased in septic mice in CLP+berberine+yohimbine and CLP+yohimbine groups ( P<0.05) .Flow cytometry analysis demonstrated the similar results in the apoptosis of splenocytes and T lymphocytes.However, only yohimbine treatment reduced the apoptosis of B lymphocytes in the spleen of sepsis-challenged mice.Compared with CLP group, caspase-9 activity was significantly re-duced in CLP+berberine group (P<0.05), the activities of caspase-3, caspase-8 and caspase-9 were all statistically re-duced (P<0.05) in CLP+yohimbine group and CLP+yohimbine+berberine group.CLP significantly increased the ex-pression of cytosolic Fas, Bim and mitochondrial Bax in the splenocytes, and decreased Bcl-2 expression compared with sham group.Compared with CLP group, the expression of cytosolic Bim and mitochondrial Bax in CLP+berberine group were reduced (P<0.05).Fas expression decreased only in CLP+yohimbine group (P<0.05).Berberine combined with yohimbine reduced the expression of cytosolic Fas, Bim and mitochondrial Bax in the septic mouse splenocytes ( P <0.05).CONCLUSION:Yohimbine reduces sepsis-induced splenic lymphocyte apoptosis in mice by inhibiting Fas expres-sion and in turn blocking both extrinsic and intrinsic apoptosis pathways.Berberine reduces Bim expression and inhibits caspase-9 activation, but not caspase-3 activation and apoptosis in the septic mouse splenocytes.Berberine combined with yohimbine reduces splenocyte apoptosis in the septic mice by inhibiting both extrinsic and intrinsic apoptotic pathways.