[Effect of Buyang Huanwut decoction on apoptosis of splenocytes in rats with sepsis]

Abstract

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) 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 after 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 CD40 B 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 expression: 11.63±5.91 vs. 30.83±16.09, P<0.05 or P<0.01). Ninety-six hours after CLP, the above indexes gradually 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.