Microcystin-LR (MC-LR) is the most common microcystins (MCs) due to its ubiquity, abundance and toxicity, and can cause oxidative stress in various organs, including the reproduction system. The aim of this study was to investigate the effect of MC-LR on oxidative stress and apoptosis of Chinese hamster ovary (CHO) cells. The viability of CHO cells was decreased after treatment with MC-LR at 5, 10, 15μg/ml, and the proliferation of CHO cells was declined at 2.5, 5, 10μg/ml MC-LR. MC-LR increased the content of MDA in CHO cells, indicating that MC-LR accelerated lipid peroxidation of CHO cells. MC-LR decreased the activity of CAT in CHO cells, indicating that MC-LR decreased the antioxidant ability of CHO cells. Moreover, the MC-LR also induced generation of reactive oxygen species (ROS), mitochondrial membrane potential loss, and cells apoptosis. Real-time PCR (qPCR) analysis of p53, bax, and bcl-2 genes revealed p53 and bax were up-regulated for about 1.5 fold, and bcl-2 was down-regulated in CHO cells after MC-LR treatment. Therefore, the acute exposure to MC-LR could promote generation of oxidative stress, depress cellular viability and cause cells apoptosis and the changes of apoptosis-related genes in CHO cells.

Objective To study the oxidative stress of Chinese hamster ovary(CHO) cells induced by microcystins-LR through testing the changes of catalase(CAT) and malondialdehyde(MDA).Methods CHO Cells were seeded at 1×105 cells per well of a 96 well-plate for 24 h.The cells were exposed to different doses of microcystin-LR(0,1,5,10 and 15 μg/ml) for 24 h.The viability of cells was determined with MTT assay.Meanwhile,the EC50 value was determined.The cells were inoculated at 1×106 cells per well of a 6 well-plate and were exposed to different doses of microcystin-LR [0 μg/ml,2.5 μg/ml(1/4 EC50),5 μg/ml(1/2 EC50),10 μg/ml(EC50)] for 24 h.Then,viability of CAT and the content of MDA were determined.Results With the increases of MC-LR concentration,CHO cell viability showed a declining curve.Compared with the control group,a significant reduce of cytoactive were seen in 5,10 and 15 μg/ml groups(P0.05).The cytoactive in 10 μg/ml group was 53.8% which closed to 50%,so EC50 was about 10 μg/ml.Compared with the control group,the viability of CAT in every MC-LR exposed group was significantly reduced(P0.05),and the content of MDA was significantly increased(P0.05).Conclusion The results in the present study suggest that MC-LR may cause oxidative damage on CHO cells.

Objective To study the effect of apoptosis induced by microcystin-LR(MC-LR) in Chinese hamster ovary(CHO) cells.Methods CHO cell density was adjusted to 1 ×105/ml,and treated with different concentrations of MC-LR(0,1,5,10,15 μg/ml) for 24 h.MTT method was used to measure cell survival rates,and the half maximal effective concentration(EC50) was calculated.After treatment with 0(control),2.5 μg/ml(1/4 EC50),5 μg/ml(1/2 EC50),10 μg/ml(EC50) MC-LR for 24 h,reactive oxygen species(ROS),mitochondrial membrane potential(MMP),caspase-3 activity,the apoptosis rate in CHO cells were measured.Results In each concentration group,intracellular ROS generation,caspase-3 activity,cell apoptosis rate increased and MMP decreased significantly compared with the control group(P0.05).In addition,with the increases of MC-LR concentration,ROS content,caspase-3 activity,the apoptosis rate in CHO cells increased and MMP decreased.Conclusion Exposure to MC-LR may induce apoptosis in CHO cell in vitro.

Objective To study the changes of apoptosis morphology and the effect of caspase-3 on the apoptosis induced by microcystin in rat Sertoli cells.Methods Sertoli cells were isolated and purified from youthful SD rats and the cell model was established.After exposure to MC-LR(0,1,10 μg/ml),apoptosis of Sertoli cells induced by MC-LR was detected using Hoechst fluorescent staining.The caspaure activity in Sertoli cells was measured by cleavage of the caspase substrate.Results The number of apoptosis Sertoli cells progressively increased with the increasing MC-LR dosage.After Sertoli cells were exposed to 10 μg/ml MC-LR MC-LR,caspase-3 activity significantly increased.Conclusion MC-LR may induce apoptosis of Sertoli cells.Caapase-3 may play an important role in the apoptosis of Sertoli cells induced by MC-LR.

Objective To study the effects of MC-LR on the levels of apoptosis related proteins in rat Sertoli cells.Methods Sertoli cells were isolated and purified from SD rats and the cell model was established.After exposure to MC-LR(0,0.5,1,10,20 μg/ml) for 24 and 48 h,apoptosis of Sertoli cells induced by MC-LR was detected.The caspase activity in Sertoli cells was measured by cleavage of the caspase substrate.And the effects on the levels of apoptosis related proteins were detected by Western blot.Results Microcystin-LR(10 and 20 μg/ml) made the cytoactive significantly decreased when Sertoli cells were exposed to microcystin-LR for 24 and 48 h.After exposure to 1 μg/ml microcystin-LR for 24 h,the level of P53 protein significantly increased,and the level of Bax and Bcl-2 protein had no significant change.When cells were exposed to 10 μg/ml microcystin-LR,the level of P53 and bax protein in Sertoli cells significantly increased,and the level of Bcl-2 protein significantly decreased.After cells were exposed to 1 μg/ml microcystin-LR for 48 h,the level of P53 protein increased but had no significant difference,the level of Bax protein significantly decreased,and the level of Bcl-2 protein significantly increased.When cells were exposed to 10 μg/ml microcystin-LR,the level of P53 and bax protein significantly increased,and the level of Bcl-2 protein significantly decreased.Conclusion MC-LR with higher doses(10,20 μg/ml) have suppression effect on cytoactive.The apoptosis related proteins of p53,bcl-2,bax are involved in apoptosis of Sertoli cells induced by MC-LR.

To study the oxidative stress effects of microcystin-LR on rats Sertoli cells, and to explore the toxic mechanisms of microcystin-LR on reproductive system. Methods: Rat Sertoli cells were isolated and cultured. Purity of extracted Sertoli cells was identified by Feulgen staining method. Viability of primary rat Sertoli cells and the maximum dose of non-cytotoxicity of MC-LR on Sertoli cells were ascertained with MTT method after the cells were treated with the different concentrations of MC-LR for 24h. LDH, MDA, SOD, ROS in Sertoli cells were analyzed after Sertoli cells were cultured with different concentrations (0 mu g/L, 0.15 mu g/L, 1.5 mu g/L, 15 mu g/L) of MC-LR for 6, 12, 24h,. Results: The Sertoli cells model was obtained to study the toxic effects of MC-LR. The highest non-toxicity concentration of MC-LR was 15 mu g/L. The level of ROS in cells increased after exposed to the different concentrations of MC-LR, and there was a statistically significant difference when cells were exposed to 15g/L compared to control cells(P<0.05). There was no statistically significant difference of MDA between control cells and cells exposed to MC-LR (P>0.05). The changes of lactate dehydrogenase (LDH) leakage amount were not significant after cells were cultured with the different concentration of MC-LR (P>0.05). The decreases of superoxide dismutase (SOD) were found to be dependent on the dose (P<0.05). Conclusion: MC-LR caused change of ROS in this study, but it had no effects on MDA. MC-LR had no effects on leakage rate of lactate dehydrogenase (LDH) but enhanced the activity of superoxide dismutase (SOD). The results suggested that MC-LR can induce oxidative stress in primary cultured rat Sertoli cell, but can not lead to lipid peroxidation. [Dan Yi, Xiaohui Liu, Fengquan Zhang, Jun Wang, Yang Zhao, Dongjie Sun, Jinwei Ren, Huizhen Zhang. Oxdative stress on Sertoli cells of rats induced by microcystin-LR. Life Science Journal. 2011;8(2):249-253] (ISSN:10978135). http://www.lifesciencesite.com.

Toxic cyanobacterial blooms in freshwater have been considered as threats to human health. Microcystins are a family of cyclic polypeptides produced by cyanobacteria and are toxic to plants and animals. Microcystin-LR (MC-LR) is the most toxic variant among the microcystin family and could cause oxidative stress in various organs, including the reproduction system. The aim of this study was to investigate the effect of MC-LR on apoptosis of Sertoli cells that play an essential role in the development and maturation of sperm cells. Sertoli cells were isolated from healthy immature rats and cultured with MC-LR. The viability of Sertoli cells was decreased after treatment with MC-LR at 10 µg/ml for 24 h (P < 0.05). Moreover, the MC-LR-treated cells exhibited condensed chromatin and fragmented nuclei, features of apoptosis, as judged by Hoechst 33258 staining. We also analyzed the mRNA and protein levels of three apoptosis-related genes, p53, bax and bcl-2, using reverse transcription-polymerase chain reaction and Western blot analyses, respectively. Both p53 and bax function as promoters of apoptosis, while bcl-2 is an apoptotic suppressor. The mRNA and protein expression levels of p53 and bax were increased in Sertoli cells treated with MC-LR at 10 µg/ml compared with the control group (P < 0.05), while the bcl-2 protein levels were decreased in cells treated with MC-LR at 10 µg/ml (P < 0.05). Moreover, caspase-3 activity that is involved in the induction of apoptosis was significantly increased in Sertoli cells treated with MC-LR. These results indicate that MC-LR induces apoptosis of Sertoli cells.

[Objective]To study the cytoactive effect and effect on apoptosis-related genes by different dose of microcystin-LR.[Methods]Extracted sertoli cells were exposed to low(0-500×10-3μg/ml)and high(1-20μg/ml)dose of microcystin-LR. Then the cytoactive effect was detected using MTT method and NR method after 24h,48h or 72h. Sertoli cells were exposed to 1 and 10μg/ml microcystin-LR for 24h and 48h respectively,and the total RNA of cells was extracted. Then the expression of P53,bax and bcl-2 were detected by using RT-PCR method.[Results]Low dose microcystin-LR increased the cytoactive of sertoli cells,but the high dose microcystin-LR inhibited the cytoactive. But the cytoactive decreased with exposed time delay. When sertoli cells were exposed to 1μg/ml microcystin-LR,microcystin-LR up-regulated P53 and bcl-2 but down-regulated bax gene expression. When sertoli cells were exposed to 10μg/ml microcystin-LR,microcystin-LR up-regulated P53 and bax but down-regulated bcl-2.[Conclusion]Low dose microcystin-LR could increase the cytoactive of sertoli cells,but microcystin-LR could obviously inhibit the cytoactive along with increasing dose and exposed time. At the same time,microcystin-LR effected apoptosis of sertoli cells through effecting P53,bax and bcl-2 gene expression.

Objective: To explore the inhibition of 22 kinds of Chinese herbs on α-glucosidase and screen Chinese herbs which have greater inhibition.Methods: The experiment was carried out with maltose as substrate,and then established optimal reaction conditions.The inhibition of 22 kinds of Chinese herbs and acarbose on α-glucosidase were tested by the enzyme reaction system.Here,acarbose was used as a positive control group.Results: All of the 22 kinds of Chinese herbs could inhibit the activity of α-glucosidase,and Sangye,Sangbaipi,Diyu,Qingguo and Wubeizi were the fi rst fi ve,the inhibition rates were 63.19%,76.55%,70.03%,78.18% and 92.83% respectively at the concentration of 500mg/ml.The inhibition of Wubeizi was the strongest,and when its concentration reached 1000mg/ml,the inhibition rate increased to 95.44%,which was similar to the effect of 5mg/ ml acarbose.Conclusion: Compared with the other Chinese herbs,Sangye,Sangbaipi,Diyu,Qingguo and Wubeizi had stronger inhibition on α-glucosidase in vitro.

微囊藻毒素（microcystins，MC）是由微囊藻等蓝藻产生的具有生物活性的单环七肽小分子化合物，在目前的检测条件下检测到80多种同分异构体［1］，其中MC-LR是最常见也是研究较深入的一个亚型。蓝藻的生长受多种因素的影响，有研究［2］。表明总氮和总磷等与水中藻类密度密切相关。