Study The Mechanism Of Cadmium-induced Autophagy And Its Effect On Apoptosis

Due to the development of modern industry,the exposure of cadmium exposed to the environment has been increasing strikingly.Cadmium mainly enters human body through the food chain,the long clearance time around 40 years.A long-term exposure to cadmium causes severe damage to many organs,such as liver,kidney,lungs,etc.and a variety of diseases such as hypertension,hyperlipidemia and so on.Biological toxicity of cadmium has been extensively studied.However,the biological effects of cadmium on spleen,an important immune organ in the human body,are scarcely studied.Therefore,we studied the mechanism of cadmium exposure induced autophagy and its effect on apoptosis.In this study,we used mice and a B lymphocyte cell line Ramos cells as models.The mice were exposed to cadmium by feeding on cadmium supplemented food continuously for a week.Ramos cells were treated by supplemented with 0-5 ?M Cd in culture medium for 24 hours.The autophagic biomarker LC3-II was increased,and P62 protein was decreased in a dose-dependent manner revealed by Western blot.The result showed the green fluorescence intensity increased in cadmium exposed cells,which demonstrated Cd increased the acid corpuscle.These results demonstrate cadmium exposure increased the autophagic activity in mouse spleen and Ramos cells.Previous study cadmium exposure resulted in VMP1 protein accumulation in spleen and Ramos cells.Vmp1 is a transmembrane protein with six hydrophobic regions,which has been shown to be required for autophagy in mammalian cells.In this study the expression of VMP1 was silenced by RNAi technology to investigate its function in cadmium induced autophagy.When the accumulation of VMP1 inhibited by siRNA,the increase of LC3-II was abolished under the exposure of cadmium,indicating the cadmium induced autophagy in Ramos cells was dependent on protein VMP1.When protein synthesis was blocked by CHX,the degradation rate of VMP1 in Ramos cells was measured by Western blotting.The result demonstrated the exposure of cadmium increased the stability of VMP1,which might contribute to the cadmium increased VMP1.It has reported that reactive oxygen species(ROS)induced autophagy.In our study,the exposure of cadmium increased the ROS and calcium level in Ramos cells.When the cells treated with ROS scavenger TCP,the induction of ROS and calcium by cadmium was abated,and the induction of autophagy was also abated.However,when Ramos cells were treated by calcium ion channel IP3 R inhibitor 2-APB,the ROS did not change significantly in presence of cadmium,but induction of calcium level was abated,and the accumulation of VMP1 and LC3-II was also abated.The results indicated the ROS mediated induction of autophagy by cadmium was dependent on the increase of calcium level and accumulation of VMP1,and the increase of calcium and VMP1 was the downstream signal of ROS in the signal transduction pathway of cadmium induced autophagy.The effect of cadmium-induced autophagy on apoptosis was investigated by Flow cytometry and Western blot.The results showed that cadmium could induce Aninxin V positive cells and the expression of apoptotic protein PARP?Caspase3 increasing.Apoptosis weakened and the number of Aninxin V positive cells was decreased after inhibiting autophagy by CQ.Lactate dehydrogenase(LDH)was released after cell rupture,Cd and CQ co-treatment decreased the LDH activity compared with cadmium solo-treatment.The results demonstrated cadmium-induced autophagy increased apoptosis in Ramos cells.