Objective To observe the change in number of spleen T lymphocyte, B lymphocyte,apoptotic splenocytes and the expression of Bcl-2 and Bax protein in rats with sepsis; to study the effect of Buyang Huanwu decoction(BYHWD,补阳还五汤)on the immune function in rats with sepsis.Methods One hundred and eighty healthy male Wistar rats were randomly divided into four groups: sham operation group (n= 30), model group (n=50), BYHWD treatment group (n=50) and BYHWD prevention group (n= 50). Sepsis model was reproduced by cecal ligation and puncture (CLP). The rats of BYHWD treatment group and BYHWD prevention group were given BYHWD (1 g/ml) 15 ml/kg at 30 minutes after surgery,and 3 days before surgery, once a day, respectively. The rats were sacrificed and their spleens were harvested at 6, 12, 24, 48, 96 hours after reproduction of the model. Morphological changes in spleen, the expression of T lymphocyte, B lymphocyte, apoptotic cells, Bcl-2 and Bax were determined in all the groups.Results By light microscopy, it was found that white pulp became atrophic and splenic nodules destroyed fter CLP. The pathology was most obvious in model group, followed by BYHWD treatment group, and least obvious in prevention group. CD4+ T lymphocyte, CD40 B lymphocyte and Bcl-2 protein expression in model group were obviously reduced compared with those of sham operation group, but the number of apoptotic cells and Bax protein expression were elevated, reaching their nadir or peak 24 hours after the surgery[the optical densities (A value): CD4+ T lymphocyte: 18. 28± 4.57 vs. 98. 60±18.18; CD40 B lymphocyte: 26. 96±6.26 vs. 104. 87±30.97; Bcl-2 protein expression: 20. 23±11.75 vs. 149. 67±5.24;apoptotic cells: 241. 75±44.79 vs. 14. 67±5.24; Bax protein expression: 128. 75±44.79 vs. 5. 34±4.26,all P＜0. 01], then they gradually increase or decrease. CD8+ T lymphocyte count did not significantly changed. The results showed that BYHWD could markedly elevate the level of CD4+ T lymphocyte and CD40B lymphocyte, and lower the protein expression of apoptotic cells and Bax. There were no significant changes in the CD8+ T lymphocyte and Bcl-2 protein expression. Furthermore, the results in the BYHWD prevention group were better than those in BYHWD treatment group (A value: CD4+ T lymphocyte: 94. 12±15.45 vs.72.37 ±8. 00; B lymphocyte: 90. 46 ± 13. 34 vs. 55.66 ± 4.23; apoptotic cells: 27.63 ± 9.91 vs. 40. 83 ±16.09; Bax protein expressoin: 11. 63±5.91 vs. 30. 83± 16.09, P＜0. 05 or P＜0. 01). Ninety-six hours after CLP, the above indexes graduallv approached to the level of the sham operation group. Correlation analysis showed that cell apoptosis and Bax were positively correlated (r= 0. 522,P=0.000), but cell apoptosis and Bcl-2 showed negative correlation(r=-0. 659,P=0.000).Conclusion BYHWD improves the immunological suppression in rats with sepsis by lowering apoptosis of CD4 T lymphocyte and B lymphocyte in spleen, and its underlying mechanism may be that BYHWD produce a decrease of apoptosis through Bax.%目的 通过观察脓毒症大鼠脾脏T、B淋巴细胞数量、细胞凋亡及Bcl-2、Bax蛋白表达的变化,探讨补阳还五汤对脓毒症大鼠免疫功能的影响及其作用机制.方法 按随机数字表法将180只雄性Wistar大鼠分为假手术组(30只)、模型组(50只)及补阳还五汤治疗组(50只)和防治组(50只).采用盲肠结扎穿孔术(CLP)制备脓毒症大鼠模型.治疗组于术后30 min、防治组于术前3 d灌胃补阳还五汤浓缩液(含生药1 g/ml)15 ml/kg,每日1次.分别于术后6、12、24、48、96 h处死大鼠,观察各组动物脾脏形态学变化,脾T、B淋巴细胞,凋亡细胞,Bcl-2、Bax蛋白表达的变化.结果 光镜下观察CLP术后大鼠脾脏白髓逐渐萎缩,淋巴小结破坏,损伤的严重程度为模型组＞治疗组＞防治组.与假手术组比较,模型组术后各时间点CD4+T淋巴细胞、CD40 B淋巴细胞、Bcl-2蛋白表达明显降低,凋亡细胞、Bax蛋白表达明显增加,均于24 h达谷值或峰值[均为积分吸光度(A)值:CD4+T淋巴细胞:18.28±4.57比98.60±18.18;CD40 B淋巴细胞:26.96+6.26比104.87±30.97;Bcl-2蛋白表达:20.23±11.75比149.67±5.24;凋亡细胞:241.75±44.79比14.67±5.24;Bax蛋白表达:128.75±44.79比5.34±4.26,均P＜0.01];而CD8+淋巴细胞无明显变化.与模型组比较,补阳还五汤治疗组和防治组各时间点脾脏CD4+T淋巴细胞、CD40B淋巴细胞明显升高,凋亡细胞、Bax蛋白表达明显减少,而CD8+T淋巴细胞、Bcl-2蛋白表达无明显变化;防治组治疗效果优于治疗组(A值:CD4 +T淋巴细胞:94.12±15.45比72.37±8.00;CD40 B淋巴细胞:90.46±13.34比55.66±4.23;凋亡细胞:27.63±9.91比40.83±16.09;Bax蛋白表达:11.63±5.91比30.83±16.09,P＜0.05或P＜0.01);至96 h时各指标逐渐接近假手术组水平.细胞凋亡与Bax呈显著正相关(r=0.522,P=0.000),与Bcl-2呈显著负相关(r=-0.659,P=0.000).结论 补阳还五汤通过减少脾脏CD4+T淋巴细胞、B淋巴细胞凋亡,从而改善脓毒症时的免疫抑制;这可能与补阳还五汤减弱Bax对细胞凋亡的促进作用相关.
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