Exploration On The Oxidative Damage Of Rat Granulose Cells Induced By Microcystin-LR And Its Mechanism

ObjectiveThe rat granulose cells were incubated in vitro and the cell proliferation, hormone secretion, DNA damage, apoptosis and the changes for the expression of Bax and Bcl-2 were observed. Consequently, we expected this study would provide a sight to the oxidative damage of rat granulose cells induced by Microcystin-LR and its mechanism.Method1. The granulose cells were extracted from 21-25 days old female Wistar rats under sterile condition and cultivated. Cell growth curve was obtained. The cells in log phase were isolated and administrated MC-LR at the dosage of 0, 10, 20 and 30μg/ml. MTT was performed to observe the proliferation of exposed cells on 24h, 48h and 72h, thus the MC-LR exposure model of rat granulose cells was established.2. After 48h exposure to MC-LR at 0, 10, 20 and 30μg/ml, fluorescence was performed to detect the level of ROS inside the cells. Spectrophotometry was applied to measure the concentration of T-SOD and MDA in the cell culture fluid.3. MTT was applied to acquire to the data of cell proliferation and ECL was performed to measure the level of estradiol(E2 )and progesterone(P) in cell culture fluid.4. Single cell gel electrophoresis(SCGE) was performed to evaluate the DNA damage of the granulose cells.5. TUNEL was applied to detect the apoptosis of cell culture fluid.6. RT-PCR was applied to detect the mRNA changes of expression of Bax and Bcl-2. Results1. After the MC-LR exposure at the dosage of 10, 20 and 30μg/ml for 48h, the cell proliferation was significantly suppressed (P<0.05). According to the result of Spearman analysis, there was a negative association between dosage of MC-LR and cell proliferation (r=-0.767, P=0.000) , and dose-response relationship was obtained. Consequently, we selected 48h as the optimal effect time of MC-LR to establish the model of rat granulose cells.2. The level of MDA and ROS in cell culture fluid was simultaneously increased with the MC-LR dosage. In contrast, the T-SOD was negatively associated with the dosage of MC-LR; Being compared with control group, the MDA and ROS concentration in cell culture fluid of 20μg/ml and 30μg/ml MC-LR group was decreased with significance (P<0.05); There were significant differences (P<0.05) in all dosage group on the activity of T-SOD when compared with control group.3. The concentration of E2 and P among 20μg/ml and 30μg/ml groups were significantly reduced (P<0.05) when compared with control group.4. The tailing cell counts increased with the dosage of MC-LR, indicating that the DNA damage advanced with the dosage.5. The cell apoptosis rate was elevated with the dosage. Compared with control group, the apoptosis rate of 20μg/ml and 30μg/ml was significantly higher.6. The cell assay indicated that the expression of Bax was up-regulated and Bcl-2 down-regulated. The difference of 20μg/ml, 30μg/ml to the control group was significant (P<0.05).Conclusions1. MC-LR is capable of inducing oxidative stress of rat granulose cells in vitro, and level of ROS was ascended. T-SOD activity in cell culture solution was significantly suppressed, in contrast the MDA was increased with dose-response relationship.2. According to the result of in vitro experiment, MC-LR has been identified to maintain cellular toxicity to rat granulose cells. With the significant suppression to cell proliferation of rat granulose cells, the cellular apoptosis rate and DNA break were also increased. The expression of Bax was up-regulated, and the expression of Bcl-2, the capacity of secreting E2 and P were all decreased with dose-response relationship.3. There is solid consistency in the oxidative stress and cellular damage induced by various doses of MC-LR in rat granulose cells, indicating the MC-LR can cause the damage of rat granulose cells by triggering the oxidative stress, consequently the apoptosis would happen. The oxidative stress caused by MC-LR may be an important and promising mechanism of cellular toxicity for rat granulose cells.