The Signal Mechanism Underlying DON Induced Apoptosis in BHK-21 Cells
|School||Nanjing Agricultural College|
|Course||Clinical Veterinary Medicine|
|Keywords||Deoxynivalenol BHK-21 cells apoptosis signal mechanism underlying|
Deoxynivalenol(DON,vomitoxin) is a trichothecene secondary metabolite produced by Fusarium species. It was wildly contaminated in raw material and their production, which have been to threaten human and animals’health. Previous studies indicated that DON has potent toxicity to many of cells, such as procaryotic cells, eucarhotic cells. It could inhibit the synthesis of proteins, DNA and RNA. It also could inhibit the function of mitochondrial and induced apoptosis. Induced apoptosis is the most significance pathway which DON exerts its pathopenicity and immunotoxicity, It also was the popular problem in research filed of mycotoxin in recent years. At present, the study mostly focus on the cytotoxicity of DON, while few are reports of the signal mechanism underlying DON induced apoptosis.Therefore, hamster renal cell BHK-21, which is sensitive to DON, was exposed with DON to study its influence to morphology, mitochondrial membrane potential and variation of expression of proteins related to apoptosis pathway. The mechanism was investigated for overall lern the toxicity and the biological action of DON.TEST1:In the present experiment, Exponential phase of growth of BHK-21 cells were exposed to five different concentration of DON(125 ng mL-1,250 ng mL-1,500 ng ml-1,1000 ng ml-1,2000 ng mL-1) for 24 h. The morphological changes of the BHK-21 cells were observed under inverted phase contrast microscope and DNA integrity were detected agarose gel electrophoresis. The result show that the different concentration of DON could induce apoptosis for BHK-21 cells, changing of cell morphology exhibited a dose-effect relationship with DON, and DNA was fragment into DNA ladder.TEST 2:In the present experiment, Adaptation of BHK-21 cell were in exponential phase of growth and were exposed to five different concentration of DON (125 ng mL-1,250 ng mL-1,500 ng mL-1,1000 ng mL-1,2000 ng mL-1) for 6 h,12 h,and 24 h, respectively. Then stained by JC-1, finally mitochondrial brane potential of changes were detected flow cytometry(FCM). The result show that BHK-21 cells exposed to DON for 24 h, a significant change of the mitochondrial brane potential of cell lines in defferent concentrations comparing with control group(P<0.05), except125 ng mL-1. Marked decreasing of mitochondrial membrane potential in a dose-dependent manner were observed. Moreover, there was a significant difference(P<0.05) between the experimental group and the control group at concentration of DON about 2000 ng mL-1, on the other hand, The mitochondrial membrane potential declined gradually with the prolonging action time.Marked decreasing of mitochondrial membrane potential in a time-dependent manner were observed. The expression of Bax, Bcl-2 and p53 after DON treated was detected with Immunocytochemical staining.The analysis showed that, after 24 h treatment with DON, the expression of Bax was significant higher, while Bcl-2 decreased.TEST 3:The expression of caspase-3、caspase-9、AIF、caspase-12 were detemined by Western blotting at treatment of different concentration of DON. The result show that the protein molecular weight of caspase-3, caspase-9, AIF and caspase-12 are 35,47,57 and 55 KD, respectively. The brown positive band could be observed in all the two groups after treated by Electrophoresis and transmembrane, and it becomes deeper gradually and the breadth is enhanced with increasing of concentration of DON. Refering toβ-actin (42KD), quantitative Analysis of hybridization bands by image analysis system, which further verifies that the treatment by DON could increase the expression of caspase-3, caspase-9, AIF and caspase-12 in BHK-21 cells.