Study On The Mechanism Of Lead Stress-induced Autophagy In Dugesia Japonica Cells

Lead is a toxic heavy metal that is harmful to human,animals and the environment.Lead pollution is widely distributed and exited in the environment and result in global threats because of lead using in industry and agriculture.At present,toxicological studies on lead stress have entered the molecular level.Studies have demonstrated that lead can cause subcellular structural disruption,oxidative stress increase,genetic material damage,and induce autophagy,apoptosis and other reactions in cells.For the moment,there are abundant researches about the autophagy induced by lead stress,however,the mechanism in which has not been fully elucidated.On account of fresh water planaria is very sensitive to the environment and distributed in a quantity of fresh water systems,planaria as toxicology model accepted by many scholars in that planaria toxicology have developed gradually in recent years.Thus,evaluation and research of lead toxicology were experimented with Dugesia japonica living in fresh water.We use Dugesia japonica as experimental animal to study the autophagy under lead stress.On condition that acute toxicology of lead(80 mg/L),the autophagy and the expression change of autophagy associated proteins LC3,ULK1 and Beclinl was detected with different lead treatment time(0,6,8,12,24,36 and 48 h).Meanwhile,The effects of Ca2+ chelating agent BAPTA-AM on the expression change of autophagy and autophagy associated protein were investigated to explain the Dugesia japonica autophagy time occurrence,signaling pathway and the mechanism under Pb2+ exposure.Meanwhile,to further detect the occurrence and development of apoptosis,autophagy inhibitor 3-MA was used to inhabit autophagy,so as to explore the relationship between autophagy and apoptosis under acute lead exposure.The main results obtained in this experiment are as follows:1.After being exposed to Pb2+ stress,the planarians showed obvious morphological changes which are mainly "C" type crimp,weak activity,the absence of photophobism and degradation phenomenon with death in some planarians.2.Observing the structure of autophagosome by transmission electron microscope.Under the lead exposure after 6h,a large number of double membrane autophagic vacuoles were observed,while after 24 h there are significant reduced number of autophagosomes and mature autophagosomes can be seen.However,autophagosomes were not observed under the lead exposure after 36 h or 48 h,suggesting that autophagy mainly happened within 24 h under the lead exposure.3.Detecting the expression of mRNA and protein of autophagy marker-LC3.The results showed that Dugesia japonica autophagy was induced by lead exposure and mainly occurred within 24 h.4.The expression level of ULK1 in Dugesia japonica cells increased significantly after exposure to lead solution for 6 and 8 h,the expression of ULK1 decreased later,but it still significantly higher than that of the control group within 36 h.However,the expression of p-ULK1 increased gradually at 6 h,8 h,and increased significantly at 8 h.Compared with the lead treatment group,the expression of ULK1 in BAPTA-AM+Pb2+co-treated group was significantly decreased when adding B APT A-AM,indicating that BAPTA-AM could significantly inhibit the expression of ULK1.5.There was no significant change in the expression of Beclinl in the cells exposed to lead solution within 24 h,while after 36 h the expression of Beclinl was significantly increased compared with the control group.Indicating that Beclinl was not involved in autophagy pathways induced by lead stress.Autophagy activation induced by lead stress may refer to mTOR/ULK1 and other downstream signaling pathways of ULK1.The expression of Beclinl in BAPTA-AM and Pb2+ co-treated group was significantly decreased compared with lead treatment group,indicating that BAPTA-AM could significantly inhibit the expression of Beclinl.6.The expression levels of cleaved caspase-8 and cleaved caspase-3 were detected by autophagy inhibitor 3-MA(5mM)inhibiting autophagy.The expression of cleaved caspase-8 were significantly increased in the treatment of 6 h,8 h,and 12 h,but there was no significant change after 24 h compared with the control group.Cleaved Caspase-3 expressional quantity increased significantly at 6 h,8 h,and the cleaved Caspase-3 expression increased at 12 h,however there was no significant change after 24 h.Conclusion:Lead exposure induced Dugesia japonica autophagy and mainly take place within 24h.Combining the researches of our lab:The autophagy induced by lead exposure mainly occurred after 36 h,suggesting that under the acute lead toxicity,Dugesia japonica cells are jointly stressed by autophagy and apoptosis.Autophagy was induced at the early age,then,cell experienced apoptosis following the increase time of lead stress and deepen toxicity.After inhibiting autophagy by inhibitors,cell apoptosis plays an important role as major approach.Autophagy mechanism induced by lead stress played a role through the mTOR/ULK1 signaling pathways,while ULK1 downstream regulatory protein Beclinl have no effect,there maybe exists other downstream proteins of ULK1 to stimulate autophagy.We also found that BAPTA-AM inhibited the autophagy induced by lead stress and inhibited the expression of autophagy related protein.