Autophagic Lysosomal Damage Induced By Swainsonine Is Protected By Trehalose In Renal Tubular Epithelial Cells

Locoweed refers to a group of toxic plants containing swainsonine and capable of causing typical neurological symptoms and pathological histological changes of locoweed poisoning in animals,mainly includes Oxytropis and Astragalus.Previous studies have shown that kidney damage is an early pathological histological change of madder poisoning disease in animals.Swainsonine（SW）,a major toxic component of locoweed,induces impaired cellular lysosomal function and impaired autophagic degradation.Trehalose,a natural non-reducing disaccharide has been shown to induced autophagy and alleviate lysosomal damage through a m TOR-independent pathway.However the protective effect and the underlying mechanism against the toxic damage of swainsonine are not clear.In view of this,in this study,the primary renal tubular epithelial cells of rats were used as the experimental model in vitro to investigate the damage of autologous lysosome in renal tubular epithelial cells and the protective effect of trehalose,in order to provide theoretical basis for the pathogenesis and prevention and control of locoweed poisoning disease.The results of the experiment were as follows:1.Swainsonine induced autophagy and led to impaired autophagic degradation in renal tubular epithelial cellsPrimary renal tubular epithelial cells were treated with different concentrations of SW medium（0,0.2,0.4,0.8 mg/m L）for 12 h,and the renal tubular epithelial cells were treated with 0.8 mg/m L SW for different times（3,6,12 h）.The vacuolar degeneration in the renal tubular epithelial cells was observed by inverted phase contrast microscopy,and the results showed a time-dependent increase in vacuolar degeneration.The changes in LC3-II and SQSTM1/p62 protein expression levels were analyzed and both were found to be time-dose-dependently increased.The above results indicate that SW can induce increased autophagy production and blocked degradation in cells with a time-dose-dependent relationship.2.Swainsonine can induce lysosome damage in renal tubular epithelial cellsPrimary renal tubular epithelial cells were treated with different concentrations of SW medium（0,0.2,0.4,0.8 mg/m L）for 12 h and 0.8 mg/m L SW for different times（3,6,12 h）.Lyso Sensor^TMGreen DND-189 and Lyso-Tracker Red was used to stain lysosomes,and the green and red fluorescence intensity was found to decrease in a time-dose-dependent manner.The protein expression level was detected by western blot and the result showed ATP6V1A、CTSD、CTSB、Lamp2 and TFEB protein was found to decrease in a time-dose-dependent manner.Analysis of ATP6V1A、CTSB、CTSD、Lamp2 and TFEB gene expression showed a time-dose-dependent increase,in contrast to protein expression.The results showed that sophorin could induce p H increase,lysosome hydrolytic ability and biogenesis inhibition of cellular renal tubular epithelial cells.The extracellularβ-galactosidase activities were detected by colorimetry and intracellular Ca²⁺concentration were detected by fluorescence staining.The results showed that high dose inhibitedβ-galactosidase activity and decreased intracellular Ca²⁺concentration.The results showed that high dose SW inhibited lysosomal exocytosis and was correlated with intracellular Ca²⁺concentration.The above results indicate that SW can induce lysosome damage in renal tubular epithelial cells.3.Trehalose alleviated the autophagy degradation disorder induced by swainsonine in renal tubular epithelial cellsThe cells were pretreated with 20 m M Tre for 12 h and then co-treated with 0.8 mg/m L SW for 12 h.The vacuolar degeneration in the renal tubular epithelial cells was observed by inverted phase contrast microscopy,and the vacuolar degeneration was alleviated by the addition of Tre.The expression of LC3-II and SQSTM1/p62 proteins was detected and found to be increased after the addition of Tre,showing a cumulative effect with SW,while the expression of SQSTM1/p62 proteins was reduced compared with that of SW.MDC fluorescence staining showed that Tre addition increased the number of fluorescent spots and showed an accumulative effect with SW.These results suggest that Tre can alleviate SW induced autophagy degradation by inducing increased autophagy production and enhanced degradation.4.Trehalose relieved the lysosome damage of renal tubular epithelial cells induced by swainsonine and promotes the activation of TFEB into the nucleusThe cells were pretreated with 20 m M Tre for 12 h and then co-treated with 0.8 mg/m L SW for another 12 h.Lysosomal staining with Lyso Sensor^TMGreen DND-189 and Lyso-Tracker Red revealed revealed that the addition of Tre alleviated the SW-induced decrease in green and red fluorescence intensity.Analysis of ATP6V1A、CTSD、CTSB、Lamp2 and TFEB protein expression levels by western blot revealed that the addition of Tre alleviated the reduction in ATP6V1A、CTSD、CTSB、Lamp2 and TFEB protein expression.The results showed that re alleviates SW induced lysosomal p H increase,hydrolase capacity and biogenesis.The results of the colorimetric assay for extracellularβ-galactosidase activities and the fluorescence staining assay for extracellular Ca²⁺concentration showed that Tre alleviated the SW-induced decrease inβ-galactosidase and intracellular Ca²⁺concentration at high doses.These results suggest that Tre alleviates SW induced exocytosis inhibition in renal tubular epithelial cells at high SW.The immunofluorescence staining of TFEB protein showed that the fluorescence intensity of SW treatment group was decreased,and the fluorescence spots were mainly distributed in the cytoplasm,while the fluorescence intensity of trehalose treatment group was enhanced,and the fluorescence spots were mainly distributed in the nucleus.The expression of TFEB protein was detected by the separation of cytoplasm and nuclear proteins.It was found that SW could induce a significant decrease of TFEB in the nucleus and cytoplasm,and Tre could induce a significant increase of TFEB in the nucleus,suggesting that trehalose could promote the entry of TFEB into the nucleus.The above results indicate that Trehalose relieved the lysosome damage of renal tubular epithelial cells induced by swainsonine and promotes the activation of TFEB into the nucleus.In conclusion,these experiments showed that SW can induce autophagy in renal tubular epithelial cells and inhibit degradation,while trehalose can alleviate the autophagy degradation disorder induced by SW by enhancing autophagy generation and degradation.SW can induce lysosome p H increase and hydrolase protein expression decrease in renal tubular epithelial cells.Trehalose can promote the entry of TFEB into the nucleus and induce the expression of TFEB protein in the nucleus,which may be the mechanism of trehalose exerting its protective effect.This study lays a basis for further understanding of the mechanism of cell vacuolar degeneration induced by SW and screening of protective agents which will be for the prevention and control of locoweed poisoning in in livestock.