Effects Of Cadmium Chloride On Autophagy, Apoptosis In The Rat Insuloma INS-1Cells

BackgroundType2diabetes mellitus （T2DM） is presently the third factor that could causedeath ranking after cardiovascular disease and cancer, and it is clear that the diabetesattack correlates with environment pollution, which is an important precipitatingfactor. As the environment pollution becomes severer for the past years, the mobidityof diabetes is also increasing. However, cadmium is a very common and representiveheavy metal in environment pollution. At present, there are so many researches onassessing the influence of cadmium on human health and the concentration-responsemodels building on different organs or systems, such as the influences of cadmium oncancer, kidney or liver damage, osteoporosis or osteomalacia, reproduction system.On contrary to that, the study between cadmium and pancreatic or diabetes is scare. Atthe same time, the research on autophagy has becomed a hot spot in recent years.There are a great quantity of researches on this field, which autophagy had included in,such as immunation, inflammation, infection, cancer, aging, and cardiovasculardiseases, diseases of liver or muscle, nerve regression. So far, the researches amongautophagy, diabetes and β cells are carrying out.But compared to those above, they aremuch less, and the specific mechanism within them has not been clarified. Toinvestigate the relationship and regulating mechanism within them is of significancein exploring clinical treatment relating medicine and providing new clues to the prevention and cure of diabetes and its complications. Our reseach use cadmiumchloride（CdCl₂）of different concentration to treat INS-1cells in vitro, then detectedthe impact of autophagy and apoptosis on INS-1cells by various kinds of experimentalmethods, which could get a thorough recognition of the impact of autophagy andapoptosis of INS-1cells treated by cadmium. Moreover, results of this study mayprovide clues to the mechanism of cadmium toxicity to the islet functions, and mayindicate new therapy targets for treating cadmium-caused islet damage.ObjectiveTo investigate the influence, precipitating factors and the underlying mechanismof the typical durable toxic pollutant cadmium chloride（CdCl₂）on autophagy andapoptosis of the rat insuloma INS-1cells, and to study the relationship between theautophagy and apoptosis induced by CdCl₂.MethodsINS-1cells at log growth phase were treated with CdCl₂, after48h used Hocheststaining to detect the cell morphological change under the microscope,Sulforhodamine B（SRB）assay was carried out to evaluate the effect of CdCl₂on cell,2’,7’-dichlorofluorescin diacetate（DCFH-DA）as fluoro-probe detecting the level ofthe intracelluar reactive oxygen species （ROS）. The autophagy markermicrotubule-associated protein light chain3-Ⅱ（LC3-Ⅱ）, the apoptosis marker polyADP-ribose polymerase（PARP）and molecular weight of70KD phosphorylatedribosomes S6kinase（p70）and its phosphorylated product p-p70{which both aredownstream proteins of （mammalian） target of rapamycin （mTOR） signalingpathway}were detected by Western bloting analysis, to find out that wether cadmiumcould induce the autophagy of INS-1cell and that is under mTOR signaling pathwayor not; The autophagy essential gene7（Atg7）was silenced via siRNA-mediatedRNAi method, then cells were detected by Western bloting analysis; INS-1cell weretreated with400μmol/L N-acetylcysteine（NAC）, and1h later cells were treated with1μmol/L CdCl₂for another48h, used SRB assay to find out whether NAC couldhave some benefits for the cell, LC3-Ⅱand cleaved PARP protein were detected with Western bloting method.ResultsAfter treated with CdCl₂for48h, morphological changes including cell shrinkage,cell size reduction and turnround were observed. Compared to the control group, theviabilities significantly decreased in0.1,1μmol/L groups, P＜0.05; as compare tothe control group, the levels of LC3-Ⅱand PARP protein of all groups both increased,between the groups the trend of LC3-Ⅱprotein presented time and dose dependence,PARP time dependence; however, the level of p70, p-p70protein has no change;compared to the negative control group, Atg7siRNA group the level of Atg7proteinsignificantly reduced; compared to negative control+CdCl₂group, Atg7siRNA+CdCl₂group the levels of LC3-Ⅱprotein obviously decreased, oppositing tothat of PARP protein; compared to the control group, ROS of all groups increased,presenting dose-dependence; compared to CdCl₂group, the viabilities of NAC+CdCl₂group increased, P＜0.05, and the levels of LC3-Ⅱand PARP decreased.ConclusionsCdCl₂may induce autophagy and apoptosis in INS-1cells by stimulating anincrease of ROS, and autophagy may be a negative regulator of apoptosis.