小檗碱与育亨宾对脓毒症小鼠脾细胞凋亡的影响及其机制研究

Effects of berberine and yohimbine on splenocyte apoptosis in septic mice

目的:探讨小檗碱与育亨宾对脓毒症小鼠脾细胞凋亡的影响及其作用机制。方法:采用盲肠结扎穿孔(CLP)构建小鼠脓毒症模型,分为假手术(sham)组、CLP组、CLP+小檗碱组、CLP+育亨宾组、CLP+小檗碱与育亨宾合剂组。CLP术后2 h灌胃给予相应药物,20 h后取脾脏,用TUNEL和流式细胞术检测小鼠脾细胞凋亡,酶荧光法检测caspase-3、caspase-8和caspase-9的活性变化,Western blotting检测凋亡相关蛋白Fas、Bim、Bcl-2和Bax的表达。结果:(1)CLP组脾脏TUNEL阳性细胞百分率显著高于sham组(P<0.05),CLP+育亨宾与小檗碱合剂组、CLP+育亨宾组凋亡细胞百分率显著低于CLP组(P<0.05)。(2)流式细胞仪检测显示CLP组凋亡的脾细胞及T淋巴细胞明显多于sham组(P<0.05),CLP+育亨宾与小檗碱合剂组、CLP+育亨宾组凋亡的脾细胞及T淋巴细胞明显少于CLP组(P<0.05)。(3)CLP+育亨宾与小檗碱合剂组、CLP+育亨宾组脾细胞caspase-3、caspase-8、caspase-9的活性均低于CLP组(P<0.05);而CLP+小檗碱组脾细胞caspase-9活性也低于CLP组(P<0.05)。(4)CLP+育亨宾与小檗碱合剂组胞浆Fas、Bim、Bax表达均低于CLP组,CLP+育亨宾组胞浆Fas表达低于CLP组,CLP+小檗碱治疗组胞浆Bim、线粒体Bax表达均低于CLP组。结论:(1)小檗碱与育亨宾合用可通过阻断内、外源性凋亡途径抑制脓毒症小鼠脾细胞凋亡,特别是T淋巴细胞凋亡。(2)育亨宾主要通过抑制Fas的表达、进而阻断内、外源性凋亡途径减少脓毒症诱导的脾细胞凋亡。(3)小檗碱可抑制脓毒症小鼠脾细胞线粒体凋亡途径,但对脓毒症小鼠脾细胞凋亡的抑制作用并不明显。

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.