The Mechanism Of JNK/MAPK Signaling Pathway On The Death Of Mouse Primary Cultured Astrocytes Induced By Cadmium Chloride

Objective:(1)Using in vitro primary cultured of cells, study the cytotoxicity of cadmium on astrocytes.(2)To investigate whether JNK/MAPK signaling pathway participate in the death of primary cultured astrocytes induced by cadmium chloride. Methods:(1)Primary cultured of astrocytes: The cerebral cortex from the brain of ICR mice in newborn 24 h was obtained, and cultured in vitro to 9~14 days. Then astrocytes were identified by GFAP immunohistochemical method.(2)The primary cultured astrocytes were exposed to 0, 5, 10, 20, 40 μmol/L cadmium chloride for 0, 3, 6, 9, 12, 24 h. The JNK specific inhibitor SP600125(50 μmol/L) pretreated cells 45 min, then giving different concentrations of cadmium chloride for 0, 3, 6, 9, 12, 24 h, and the inverted contrast microscope was used to observe the changes in cell morphology.(3)The primary cultured astrocytes were exposed to 0, 5, 10, 20, 40 μmol/L cadmium chloride for 9, 12 h, the toxicity detected by MTT assay. Cell apoptosis analyzed by Flow cytometry stained with Annexin V-FITC/PI.(4)The primary cultured astrocytes were exposed to 0, 5, 10, 20, 40 μmol/L cadmium chloride for 9, 12 h respectively. 0, 10, 20 μmol/L cadmium treated astrocytes for 0,3, 6, 9, 12 h, Western-blot was used to measure the phosphorylation level of JNK. With JNK specific inhibitor SP600125(50 μmol/L) pretreamentt of astrocytes 45 min, given different concentration of cadmium chloride for 9,12 h, the changes of JNK phosphorylation level detected by Western blot. Results:(1)Primary cultured astrocytes in vitro were polygonal, cone, etc. Cell body large, abundant cytoplasmic process, the round or oval nucleus which leaned to one side. The primary cultured astrocytes to 9~14 days in vitro were identified by immunocytochemical staining with anti-GFAP, the cells with positive immunocytochemistry were stained brown in their cytoplasm and processes. The positive immunocytochemistry of the astrocytes were counted more than 95 percents of all cells, can be used for continuous experiments.(2)Results of cell morphological changes under Inverted contrast microscope: The cell morphology have no obviously changes in 0~5 μmol/L cadmium chloride. But, it was found the cell dendritic became shorter and thinner treated with 10 μmol/L cadmium chloride. The greater damage to the shape at the concentration of 20 μmol/L cadmium chloride: Cell protruding vanished or cell becoming burr shape, the cell gap increased, sparse cells, some of the cells shrink into a spherical, necrosis, takeoff the wall and floated in culture medium. After treatment of 40 μmol/L cadmium chloride it was found cell bodies smaller and smoother, the cells becoming circular, necrosis, and the cells take off the wall and cells body floating in the culture medium. SP600125 pretreatment of 45 minutes, the change of cell morphology: 0~10 μmol/L cell morphology did not change. 20 μmol/L, cell morphology compared with Cd group: A small number of cell process become shorter and thiner, the cell body retraction. 40 μmol/L, cell morphplogy compared with Cd group: Cell processes are still connected, cell body becoming circular, but did not take off the wall.(3)MTT assay showed that cadmium chloride had a certain cytotoxicity on primary cultured astrocytes: The survival rates of astrocytes were no decreased, respectively after 0 μmol/L cadmium chloride treatment for 9, 12 h. Astrocytes was exposed at the concentrations of 5,10,20,40 μmol/L cadmium chloride for 9, 12 h, the survival rate of astrocytes decreased significantly with the increasing concentration in comparison with those in control group, the difference was statistical significant(P<0.01). Results of apoptosis rates were detected by Flow cytometry stained with Annexin V-FITC/PI: The rate of cell apoptosis in the groups treated with 0, 5, 10, 20, 40 μmol/L cadmium chloride for 9 h were(7.40±0.50)%,(9.60±0.20)%,(11.50±1.00)%,(21.70±1.10)%,(3.80±0.40)%. And for 12 h the apoptosis rate were(6.90±0.30)%,(11.50±1.50)%,(13.00±1.50)%,(16.80±1.00)%,(0.30±0.05)%, compared with the control group, there was statistical significant(P<0.01). To 40 μmol/L, along with the prolongation of time, the number of necrotic cells increased, respectively(53.40±0.25)%,(96.1±0.15)%.(4)Western blot results showed: The phosphorylation level of JNK was increased significantly as the dose increased(10~40 μmol/L)when compared to the control group(P<0.01). The phosphorylation level of JNK was increased significantly with the time increased(3~12 h)when compared to control group(P<0.01). JNK phosphorylation levels were decreased after SP600125 pretreated cells in vitro, there was a significant difference compared with Cd group(P<0.01). Conclusion:(1)In vitro primary cultured of mouse cerebral cortex astrocytes succeed.(2)Cadmium chloride has a noticeable toxic effect on mouse cerebral cortex primary cultured astrocyte, can inhibit the growth of the cells; And induced apoptosis and necrosis of astrocytes in primary cultured mouse cerebral cortex.(3)JNK/MAPK signaling pathway is involved in cadmium induced astrocyte death. JNK signaling pathway specific inhibitor SP600125 has a protective effect on cadmium induced astrocyte death.