| ObjectiveIn recent years,because of the serious environment pollution,huge social pressure and improper living habits the incidence of infertility patients has raised significantly,and especially male infertility rate is increasing year by year which influences human health and procreation.Environmental pollution is an important reason for this phenomenon.A range of environmental pollutants can lead to reproductive disorders and heavy metal is one of them.Manganese is one of trace elements which are necessary for flora,fauna and humanity.It participates in many important biochemical reactions and plays an important role in maintaining life activity of human.But simultaneously it is also a kind of metallic toxicant. Manganese and its compounds are in wide use,which are extensively used in iron and steel industry,decorative works and underground engineering support of the anti-corrosion.Its compounds are important raw materials in chemical,medicine, welding,painting,synthetic industry and so on.Nowadays,the wide application of antiknock additive Methylcyclopentadienyl Manganese Tricarbonyl,fungicide Maneb and eikonogen MnDPDP in the tomography of nuclear magnetic resonance increase opportunities of long-term and wide range exposure of Manganese and its compounds in daily life.Therefore,people begin to pay more attention to manganese toxicity. Having a certain understanding of manganese toxicity on nerve and immune system, in recent years people become to pay more and more attention to reproductive toxicity and have done a lot of work.Current studies suggest that manganese can cause toxicological effects in male reproductive system obviously.Excessive ingestion of manganese would lead some changes of pathology and enzymology in testicle organization.Research showed that manganese can result in ROS accumulation in testicular tissue by lowering the activity of testicular antioxidant enzyme.Study also showed that manganese may induce rat spermatogenic cells apoptosis with relation to down-regulation of mRNA expression of Bcl-2.Studies showed that manganese can cause disturbance of calcium homeostasis,mitochondrial dysfunction and arresting of the cell cycle,thus induce apoptosis of primary Leydig cells and reduce steroidogenesis.Whether or not apoptosis of Leydig cells induced by manganese is concerned with apoptosis-related genes is still unknown.This study will use MA-10 mouse Leydig tumor cells (mLTC-1) as a model,and observation the adverse effect of manganese chloride on the mouse Leydig Tumor cells proliferation and apoptosis through measuring the level of Bcl-2,Bax and Caspase-3 expression,it will provide a theoretical basis for the mechanisms of reproductive and developmental toxicity in the future.Methods1.The cell viability of mLTC-1 affected by MnCl2:The mLTC-1 cells of logarithmic phase were plated in 96-well plates.A:The cells were treated with manganese chloride at concentrations of 10-7,10-6,10-5,10-4 and 10-3 mol/L for 24h;B: The cell was treated with manganese chloride at concentrations of 0.1,0.3,0.5,0.7and 0.9 mM for 12h,24h,36h and 48h.The control group was left untreated.Each concentration for six parallel way,the OD value was measured by MTT.2.The situ detection of apoptotic mLTC-1 cells with TUNEL assay:The mLTC-1 cells of logarithmic phase were cultured on 6-well plate which contains coverslips at a density of 8×104 cells/well.After a 24h exposure to manganese chloride at concentrations of 0.3,0.5 and 0.7 mM,apoptotic cells were detected via TUNEL assay. 3.The mRNA level of Bcl-2,Bax and Caspase-3 in mLTC-1 cells was determined by semi-quantitative RT-PCR:The mLTC-1 cells which covered around 80%of the culture flask were treated with manganese chloride at concentrations of 0.3,0.5 and 0.7 mM for 24h.Total RNA was isolated from mLTC-1 cells,then the mRNA level of Bcl-2,Bax and Caspase-3 was measured by RT-PCR according to the manufacturer's instruction.Results1.The results of MTT assay showed that:A:manganese chloride had no remarked effect on the viability of mLTC-1 cells at the concentrations of 10-5,10-6 and 10-7mol/L,but could inhibit cells growth at the concentrations of 10-3 and 10-4mol/L and the differences was statistically significant compared with the control group(P＜0.05).B:After exposure to different concentrations of manganese chloride at 24h inhibition ratios of mLTC-1 cells were greater than them at 12h significantly, the differences had statistically significant(P＜0.05).But there had no statistically significant differences among 24h,36h and 48h(P＞0.05).2.The result of TUNEL assay:TUNEL-positive cells were shown to be stained dark brown under the light microscope and nuclear condensation was observed. Apoptotic indexs were high after treated with 0.3,0.5 and 0.7 mM manganese chloride and the differences were statistically significant compared with the control group(P＜0.05).At the concentrations of 0.5 and 0.7 mM apoptosis indexs were higher than at 0.3 mM and the differences were statistically significant(P＜0.05);At the concentration of 0.7 mM apoptosis indexs were higher than at 0.5 mM and the differences were statistically significant(P＜0.05).3.Effect of manganese chloride on mRNA Levels of Bcl-2,Bax and Caspase-3: Semi-quantitative RT-PCR results showed that the level of Bcl-2 mRNA in the mLTC-1 cells was decreased after treated with 0.3,0.5 and 0.7 mM manganese chloride and the differences are statistically significant compared with the control group(P＜0.05).The level of Bax and Caspase-3 mRNA was increased and while the differences are statistically significant compared with the control group(P＜0.05). ConclusionThis study demonstrated that manganese chloride had significant toxicity effect and can inhibit mLTC-1 cells growth and proliferation at the concentration of 10-4 mol/L,and effectively inhibit the mRNA expression of Bcl-2 and up-regulate the mRNA expression of Bax and Caspase-3.Therefore manganese chloride-induced mLTC-1 cells apoptosis may be responsible for the down-regulated expression of Bcl-2 and the up-regulated expression of Bax and Caspase-3. |
PDF Full Text Request