There was no obvious changes of astrocyte integrase morphology in the riluzole control group. The pretreatment of riluzole dramatically prevented cell morphological deterioration induced by Mn exposure. The changes of cell cycle progression were shown in Fig. 4 and Table 1. Compared to the untreated cells, the percent of G0/G1 phase cell increased 5.9, 9.28, and 11.08%, the percent of S phase cell decreased 40.69, 49.51, and 60.81%, the percent of G2/M phase cell decreased 2.59, 14.37, and 16.64%, after exposure to 125, 250, and 500 M MnCl2. Comparing with the control group, there was no obvious changes of cell cycle progression in the riluzole control group. When compared with the 500 M MnCl2 group, the percent of G0/G1 phase cell decreased 10.7%, the percent of S phase cell increased 2.07 times in the riluzole pretreatment group. Effects of MnCl2 Exposure and Riluzole Pretreatment on Apoptosis We assessed the mechanism of cell death using Annexin PKC Pathway V/PI double staining flow cytometry analysis. As shown in Fig. 5, astrocytes were exposed to 0 500 M MnCl2 for 24 h and pretreated with 100 M riluzole for 6 h.
Compared with the control group, the apoptotic P2X receptor rate increased significantly after exposure to 125, 250, and 500 M MnCl2. Comparing with the control group, there was no obvious changes of apoptosis in the riluzole control group. When compared with 500 M MnCl2 group, apoptotic rate was inhibited to 21.073.25% in the riluzole pretreatment group. As a constituent of many enzymes involved in protein and fat metabolism, manganese exhibits diverse effects in physiology. However, chronic excessive Mn exposure to this metal provokes profound neurotoxic manifestation. It is associated with a variety of psychiatric symptoms and motor symptoms. However, the mechanisms ofmanganism had not been well established. In this study, we used the cultured astrocytes to investigate that Mn exposure induced cytotoxicity, cell cycle arrest, and apoptosis on them. As shown in Fig. 1, MnCl2 could significantly suppress astrocyte viability in vitro using the MTT assay. The results demonstrated that MnCl2 inhibited cellular growth in aconcentration dependent manner. After treatment with 500 M MnCl2, cell viability was decreased to about half of nontreated control group at 24 h. This cytotoxic concentration of Mn was relatively high compared to those of heavy metals. In this study, we also measured the leakage of LDH from damaged AV-412 cells to culture media as well as the reduction of MTT.
The results also revealed that MnCl2 exposure caused a concentration dependent leakage of LDH. At the level of 500 M Mn exposure for 24 h, the leakage of LDH increased over twice than the control. The results were similar with the study of Lee et al. They found that manner significant cells death was caused by 250 and 500 M MnCl2 exposure for 24 h. However, Yin et al. found that the peaking of MTT reduction and LDH leakage occurred between Mn exposure for 2 h and 6 h that was different with our exposure time period. In addition, mancozeb, manganese ethylene bis dithiocarbamate with zinc salts, was observed to cause cytotoxicity on the cultured astrocytes. It was also shown that exposure to Mn at a concentration of either 500 M or 1 mM Mn elicited a dose dependent increase in the LDH release of the N9.