The Role Of Autophagy In The Injury Of Renal Tubular Epithelial Cells By Swainsonine In Mice

Locoweed commonly refers to any plant,usually plants from genus Astragal and Oxytropis,which produces swainsonine(SW).Currently,locoweed has been found throughout the World and has become the major toxic plant affecting the livestock production in the grassland.The estimated economical loss was 20 to 100 million dollars every year in America and China,severely hampered the development of the grassland.Swainsonine,an indolizidine alkaloid,is the principal toxic component of the locoweed which resembles mannose in chemical structure and has high affinity to the α-mannosidase and is a competitive inhibitor for α-mannosidase II leading to the accumulation of the oligosaccharides and interference of the protein glycosylation.Consumption of locoweed by the livestock may cause toxic responses characterized by symptoms of the nerve system,reproductive system,and in some cases may cause death.The extensive vacuolar degeneration is major pathological manifestation.Autophagy is a process in which cells transport the damaged,denatured and aged macromolecules/organelles into lysosome for degradation.Meanwhile,autophagy is also a biological process that pathogens invaded into body were transported and digested in the lysosome.It has been shown that interference with the processes of autophagy leads topathological vacuolar degeneration.Based on this,we used mouse TCMK-1 cell line model to investigate effects of the SW on the autophagy related proteins and to delineate the mechanism of the SW-induced toxic responses in the TCMK-1 cells in order to manage the intoxication by the locoweed.The main investigations and results are as follows:1.Swainsonine induces autophagy in TCMK-1 cellsIn order to reveal the effect of SW on autophagy in TCMK-1 cells,autophagy marker protein expression was detected by Western blot.The TCMK-1 cells were cultured with various doses of SW(0,200,400,800 μg/m L)for 12 h or were incubated with 800 μg/m L SW for 0,3,6,12 h.The expression of LC3-II and Beclin1 were significantly increased in the treated cells and these effects were dose-and time-dependent.Furthermore,when the TCMK-1 cells were stained with autofluorescent compound monodansylcadaverine(MDC),the accumulation of MDC was strikingly induced by the SW at the concentration of 400 and 800 μg/m L.Compared with the control group,the result of immunofluorescent staining of LC3 showed that LC3 levels markedly increased under the treatment of LC3 at the concentration of 400 and 800 μg/m L.In addition,the TCMK-1 cells treated with SW(400 and 800 μg/m L)for 12 h showed significantly increased autophagic vacuoles accumulated in cytosol compared to control determined by transmission electron microscopy.The above results showed that SW could induce a large number of autophagic vacuoles.The formation of the autophagic vacuole in the initiation stage of autophagy and the blocking of the following fusion with lysosome could both increase the expression of LC3-II.To further investigate the role of SW in increasing expression of LC3 protein,we co-incubated the TCMK-1 cells with chloroquine(CQ),an autophagy inhibitor,which blocks the last steps of autophagic degradation,as a result,enhance the accumulation of LC3-II.Our result showed that both SW and CQ could significantly increase the expression of LC3-II,and CQ had an additive effect with SW in inducing LC3-II,suggesting that SW promote autophagy.2.Swainsonine blocks the autophagic degradationP62 is also known as the marker protein of the autophagic activity and it connected LC3-II and ubiquitinated substrates to be degraded which will find its way to lysosome for final degradation Surprisingly,our result showed that the expression of p62 was dose-dependent and time-dependent increased under the treatment of SW in TCMK-1 cells.Subsequently,q RT-PCR experiment was performed and the results indicated that does-dependent up regulation of LC3-II m RNA expression after SW treatment for 12 h and there was no significant increases in p62 m RNA expression.These results suggested that SW caused the blockage of the autophagic degradation.The blockage of the autophagic degradation may be due to either the inhibition of the fusion between autophagosome and lysosome or the disruption of the lysosomal degradation function.We found that LC3-II and LAMP-2 fluorescent foci co-localized by immunofluorescent microscopy in SW-treated TCMK-1 cells as well as in the serum starved group except CQ-treated group.We also found numerous lysosome accumulated around the autophagic vacuole with small number of autophagosomes.3.Swainsonine induces autophagy via the inhibition of PI3K/Akt/m TOR axis in TCMK-1 cellsIn order to investigate the role of PI3K/AKT/m TOR signaling pathway in the regulation process of TCMK-1 cells autophagy by SW,Western blotting were applied to detect the phosphorylation levels of PI3 K,AKT and m TOR protein.Treatment with SW(0,200,400,800μg/m L,12 h)resulted in dose-dependent decreases in the protein levels of p-PI3 K,p-Akt,pm TOR.To further investigate the pathway effects we have analyzed the down-stream activation status of the pathway and found p-p70S6 K,p-4EBP1 levels also decreased by SW treatment and the total protein levels remained unchanged suggesting that this pathway was activated.Collectively,these results suggest that PI3K/Akt/m TOR pathway play an important role in the SW-induced autophagy.The above results showed that swainsonine could induce autophagy in TCMK-1 cells,and blocked the autophagic degradation.However,the fusion between autophagosome and lysosome was not affected,suggesting blockage of the autophagic degradation maybe due to the inhibition of the lysosomal functions.In addition,PI3K/Akt/m TOR signaling pathway is involved in the regulation of swainsonine-induced autophagy.This study provides a theoretical basis for the further research of the toxic mechanism of swainsonine.