Swainsonine Induced Cytotoxicity Of BRL-3A Cells And Antagonistic Effects Of "Jifang E" And Fe²⁺

Swainsonine (SW) is the main toxic ingredient of locoweeds, which can induce the toxication and death of livestocks and bring huge economic losses to the herdsmen in pastoral areas of China. SW can specifically inhibit the activity of Lysosomal a-mannosidase (LAM) and Golgi a-mannosidase ?(GM ?), and as a result, accumulation of oligosaccharides and abnormal prcessing of N-glcan of glycoprotein occur in the animal cells, and thus toxication occurs. So far there is no efficient antidote against swainsonine. So to investigate the toxicity and the toxic mechanism of swainsonine towards heptacytes and look for a suitable antidote against swainsonine, I carried out several researches on a normal rat liver cell line, BRL-3A, as follows:1. Effects of swainsonine on the survival rate of BRL-3A cells. The cells were treated with 100,300,500,700,900,1100 ?/mL SW for 12,24,48,72 h respectively, and the survival rate was detected. Results show that, swainsonine can reduce the survival rate of BRL-3A cells significantly, in an obvious time and dose dependent manner. The IC50 at 48th hour was 907 ?g/mL.2. Lesions of BRL-3A induced by SW. The cells were treated with 700,900,1100 ?g/mL SW for 24,48,72 h respectively, followed by dectecting the activity of LDH, ALT and AST in the cell-free medium, and the morphological observation. Results show that swainsonine can cause severe morphological changes in BRL-3A, and the cells also showed vacuolation in cytoplasm. With the increase in dose and the pass of time, the cells showed lesions like swelling or pyknosis, finally they turned to round, and fell off from the bottom. Moreover, swainsonine can cause the increase of the release of LDH, AST, and ALT in a dose-effect mode. Compared to control group, the activity of LDH, ALT and AST in the cell-free medium increase, and the differences were significant or extremely significant (P< 0.05 or P< 0.01) after treated by 900 or 1100 ?g/mL SW for 24,48 and 72 h. And the release of ALT and LDH increased significantly after the cells were treated by 700 ?g/mL SW for 48 h and 72 h (P< 0.05 or P< 0.01). All the results indicate that SW has a cytoxity towards BRL-3A cells.3. The mechanism of the toxicity of SW toward BRL-3A. After treated by swainsonine, the AMAN activity was detected through a substrate assay, and the mRNA and protein expression of MAN2A1 and MAN2B1 were detected through Realtime qPCR and Wstern blot assays. Results show that SW can cause an obvious decrease of AMAN activity in a time-dose dependent mode. Compared to the control group, at the 24th hour, only 1100 ?g/mL SW can lead the significant decrease of AMAN activity (P< 0.01), while at the 48th and 72nd hour, all the three doses of SW can lead to the significant decrease of AMAN activity (P< 0.05 or P< 0.01). Moreover, swainsonine can downregulate the gene and protein expression of MAN2A1 and MAN2B1 of the cells (P< 0.05 or P< 0.01), also in a time-dose dependent mode SW, and the downregulate effect of SW on MAN2B1 is stronger than MAN2A1.The antagonistic effects of "Jifang E" and Fe2+against swainsonine. BRL-3A cells were incubated with 10 ?g/mL "Jifang E" or 0.1?g/mL Fe2+ for an hour followed by treated with 700?g/mL SW, then the survival rate, cellular morphology of the cells, and the activity of ALT, AST and LDH in the cell-free medium, as well as, the AMAN activity and the mRNA and protein expression of MAN2A1 and MAN2B1 were dectected. Results show that, compared with the cells treated by SW alone, "Jifang E" could significantly increase the survival rate of BRL-3A at 24th and 72nd hours (P< 0.05 or P< 0.01), while, Fe2+seemed having no effects on the survival rate of SW-treated cells. Also "Jifang E" could obvious made cells recover from the lesions caused by SW to some extent at the 48th and 72nd hours, while, Fe2 had no effects. Moreover "Jifang E" can reduce the release of LDH of SW-treated cells at 48th and 72nd hours (P< 0.05), and reduce the release of ALT and AST at 48th and 72nd hours and 72nd hour respectively (P< 0.01). While Fe2+had no effect on the activity of LDH, AST or ALT in the cell-free medium. These results indicate that "Jifang E" has an obvious antagonistic effect against SW, while Fe2+seems having no antagonistic effect against SW. Also, as we hypothesized, the results show that "Jifang E" can increase the AMAN activity of SW-treated cells (P< 0.01). However, it seemed having no obvious effects on the mRNA and protein expression of MAN2A1 and MAN2B1. This indicate that the antagonistic effects of "Jifang E" against SW may worked through the mechanism of enhancing the AMAN activity.We conclude that SW can induce the lesions and decrease of AMAN activity of BRL-3A cells, and can downregulate the expression of MAN2A1 and MAN2B1, "Jifang E" can obviously protect cells from SW, and increase the activity of AMAN, while Fe2+have no obvious prtect effects.