Influences of Buyanghuanwu decoction and its decomposed formulas on cell apoptosis and expressions of Bcl-2 and Bax protein in rats with cerebral ischemia/reperfusion

Yunke ZHANG

ABSTRACT

BACKGROUND:

Buyanghuanwu decoction is a kind of commonly used Chinese herb in clinic for ischemic cerebrovascular disease, but its mechanisms have been unknown.

OBJECTIVE:

To observe effect of Buyanghuanwu decoction and its decomposed formulas on the levels of nerve apoptotic cells and expressions of Bcl-2 and Bax of brain in the ischemia/reperfusion model of SD rats and its mechanisms, study the significance of formula compatibility.

DESIGN:

Randomized controlled animal experiment.

SETTING:

Department of Neurology, First Affiliated Hospital, Henan College of Traditional Chinese Medicine.MATERIALS The experiment was performed in 3rd Grade Laboratory of State Administration of Traditional Chinese Medicine, Henan Academy of Chinese Medicine. Totally 60 clean-grade SD rats aged 4-5 months, male and female (half and half), with the body mass of 260-310 g in females and 280-330 g in males were employed in the experiment, provided by Shanghai SIPPR/BK Experimental Animal Co., Ltd. In blood activation group, 120 g danggui (Radix Angelicae Sinensis), 60 g taoren (Semen Persicae), 60 g chuanxiong (Szechwan Lovage Rhizome) were selected, and then water 1 440 mL was added and volatile oil was extracted for reserving. 120 g chishao (Radix Paeoniae Rubra), 60 g dilong (Lumbricus), 60 g honghua (Flos Carthami) and 1 440 mL water were added to the gruffs and extract, and then filtered after 1 hour decoction, added 2 880 mL water to the gruffs again, decocted for 1 hour and filtered. The decocted liquid was merged and condensed to 1 200 mL, and then we get it by adding volatile oil and tween -80 3 mL and misce bene. In huangqi (Radix Astragaliseu Hedysary) group, 1 200 g huangqi was selected and 7 200 mL water was added, decocted twice, every time for one hour, and then filtered and the decoction was merged, concentrated to 600 mL, obtained by addition of tween-80 2 mL. In Buyanghuanwu decoction group, 60 g danggui (Radix Angelicae Sinensis), 30 g taoren (Semen Persicae), 30 g chuanxiong (Szechwan Lovage Rhizome) were selected, 720 mL water was added, and volatile oil was extracted for reserving. 1 200 g huangqi (Radix Astragali),60 g chishao (Radix Paeoniae Rubra), 30 g dilong (Lumbricus), 30 ghonghua (Flos Carthami) and 920 mL water were added in the gruffs and extract, and then filtered after 1-hour decoction. 8 640 mL water was added in the gruffs again, decocted for 1 hour and filtered, and then the decocted liquid was merged and condensed to 600 mL, then we get it by adding volatile oil and tween -80 3 mL and misce bene (huangqi group and blood activation group was the decomposed formulas of Buyanghuanwu decoction group). The 1 mL drug in blood activation group was equal to 0.4g raw herbs, 1 mL huangqi (Radix Astragali) in the huangqi group to 2 g raw herbs, and the drug in the general formula group to 2.4 g raw herbs.

METHODS:

A total of 60 SD rats of clean grade were randomly assigned into sham operation group, model group, blood activation group, huangqi group and general formula group with 12 in eachgroup. The medicine was applied by gastric perfusion at the dosages of 8 g/kg, 40 g/kg and 48 g/kg in blood activation group, huangqi group and general formula group successively; and 20 mL/kg saline was applied in both sham operation group and model group. The models of cerebral ischemia/reperfusion were prepared by four vessel ligature method. Apoptotic cells were determined with TDT-mediated dUTP biotin nick ending labeling (TUNEL). Expressions of Bcl-2 protein and Bax protein were measured with immunohistochenical method.MAIN OUTCOME

MEASURES:

①Comparison of apoptotic levels of neurons in brain tissues of SD rats in each group; ②Comparison of expressions of Bcl-2 and Bax positive cells in neurons of brain tissue of SD rats in each group.

RESULTS:

①Comparison of neuron apoptotic level of brain tissues in SD rats of each group Compared with the sham operation group, the apoptosis increased in other groups (t=6.07-11.70,P＜0.01). Compared with the model group, the apoptosis decreased in the blood activation group and huangqi group (t=2.71, 2.06, P＜0.05), and decreased obviously in the general formula group (t=5.34, P＜0.01 ). ②Comparison of Bcl-2 and Bax positive cells in the neurons of brain tissues in SD rats of each groupCompared with the sham operation group, the expression of Bcl-2 staining positive cells increased in the blood activation group, huangqi group and general formula group (t=4.59-8.82,P＜0.01 ), and the expression of Bax positive cells became very significant (t=4.59-8.55,P＜0.01 ). Compared with the model group, the expression of Bcl-2 staining positive cells increased in the blood activation group, huangqi group and general formula group (t=3.48-7.75,P＜0.01 ), and the expression of Bax positive cells decreased (t=3.83-5.88,P＜0.01 ). There was no significant difference between the blood activation group and huangqi group (P＞0.05). There were significant differences in the drug effect of general formula group with huangqi group and blood activation group (P＜0.01).

CONCLUSION:

Buyanghuanwu decoction and its decomposed formulas act on being against the cerebral ischemia/reperfusion injury achieved by inhibiting cell apoptosis. Synergism of huangqi group and blood activation group leads to the strongest curative effect of general formula group.