Mechanism Of Swainsonine-induced Apoptosis In BRL-3A Cells

Swainsonine(SW) is the main toxic ingredient in poisonous plants of the genera Astragalus and Oxytropis Leguminosae plants in western China. Livestock appear classical Locoism symptoms after grazing locoweeds with extensively vacuolar degenerated cells distributing in the whole body. As is shown in the previous study, SW decreases the survival rate of BRL 3A cells, damages the hepatocytes seriously and makes hepatocytes release enezymes because of toxic damage, all of which cause the death of hepatocytes. However, the mechanism of inhibition of SW on BRL-3A has been unclear. In order to explore this problem, this present study focuses on the mechanism of SW induced apoptosis in BRL-3A cells.1. The morphological detection of apoptotic nucleus in SW-poisoned BRL-3A cells. After treating BRL-3A with different doses of SW(700, 900 and 1100 μg/mL)or with 900 μg/mL SW at different time(24, 48 and 72 h), Hoechst 33258 had been employed to dye cell nucleus in BRL-3A. The morphological changes of cell nucleus could be observed by fluorescence microscope. Results showed that, compared with control, SW causes some classical apoptotic events, such as pyknotic chromatin, deepened dyeing and fragmented nuclear and so on. The appearance of apoptosis follows time and dose dependent manner. This study suggested that SW can induce apoptosis in BRL-3A from the perspective of morphology.2. The Annexin V-FITC/PI double staining of SW-induced apoptosis in BRL-3A cells. After treating BRL-3A with different doses of SW(700, 900 and 1100 μg/mL)or with 900 μg/mL SW at different time(24, 48 and 72 h), FCM had been used to detect the apoptotic rate of BRL-3A with Annexin V-FITC/PI double staining. Results showed that, compared with control, SW causes the significant increase of apoptotic rate in BRL-3A following time and dose dependent manner(P﹤0.05 or P﹤0.01). Meanwhile, SW decreases the normal cell rate significantly(P﹤0.05 or P﹤0.01). It suggested that SW can induce apoptosis significantly in the time and dose dependent manner. The study demonstrated that SW can induce apoptosis in BRL-3A from the prospective of biochemistry.3. The expression of apoptotic proteins in SW-treated BRL-3A cells. Different doses of SW(700, 900 and 1100 μg/mL)at 48 h or 900 μg/mL SW at different time(24, 48 and 72 h) have been used to treat BRL-3A and Western blot has been utilized to detect the expression of apoptotic proteins in BRL-3A. Results showed that in comparison to control, the expression of caspase 3, 8 and 9 increases markedly, while procaspase 8 experiences decline significantly(P﹤0.05) following time and dose dependent manner. Furthermore, Fas and FasL, as well as the value of Bax/Bcl-2, see significantly increasing expression(P﹤0.05), which follow time and dose dependent manner. The result suggested that SW can induce apoptosis involved with death receptor and mitochondria pathways.