| Locoweed mainly includes poisonous plants of the genus Astragalus,Oxytropis and Sphaerophysa,and is used to describe the symptoms of "madness" in animals.Swainsonine,the main toxic component of Locoweed,is an indolizidine alkaloid,which was first isolated from Swainsona canesces and produced by endophytic fungi living in plants.With the frequent occurrence of livestock poisoning incidents in natural grasslands,swainsonine has also been found in many non-leguminous plants.The clinical manifestations of poisoned livestock are ataxia,weight loss,abortion,and even death,and the pathological changes are mainly extensive vacuoles.Therefore,these plants that contain swainsonine and can cause typical neurological symptoms and pathological changes in animals are collectively referred to as Locoweed.At present,locoweed has been found all over the world.With the disturbance of factors such as global climate change and overgrazing,it has become the main poisonous plant that threatens livestock production in natural grasslands.According to statistics,only in America and China,the annual economic loss caused by locoweed poisoning is as high as 20 million to 200 million dollars,which seriously hinders the sustainable development of animal husbandry.Therefore,clarifying the molecular pathogenic mechanism of swainsonine poisoning is the key to preventing and controlling locoweed disaster and turning poison into profit and waste into treasure.O-GlcNAcylation is a special protein post-translational modification(PTMs).A large number of studies have shown that O-GlcNAcylation can affect the physiological and pathological development of the body by regulating the process of autophagy,which is a degradation pathway necessary for the body to maintain cellular and tissue homeostasis.Previous studies have found that the clinical manifestations of animals poisoned by eating locoweed are ataxia,weight loss,miscarriage,and even death.Pathological changes are widespread vacuolar lesions in brain,liver,kidney,testis and other tissues.Its mechanism of action is that swainsonine can inhibit the activity of α-mannosidase in the body,leading to the obstruction of intracellular glycosylation modification,resulting in the accumulation of glycoconjugates such as oligosaccharides,glycoproteins,and glycolipids in cells,and finally causing the animal parenchyma is damaged,especially the central nervous system and the kidneys.Therefore,in order to further study the specific molecular mechanism of swainsonine-induced nephrotoxicity,the primary rat renal tubular epithelial cells were used as the research model to explore the molecular mechanism of autophagy mediated by O-GlcNAcylation in the injury of primary rat renal tubular epithelial cells induced by swainsonine.The results obtained in this study are as follows:1.Swainsonine induces apoptosis and autophagy in primary renal tubular epithelial cells in rats.Rat primary renal tubular epithelial cells were isolated by collagenase digestion.After treatment with swainsonine,the chromatin of the cells was pyknotic and irregular.Western blot results showed that the expression of apoptosis related proteins increased in a time-dose-dependent manner.Moreover,flow cytometry results showed that swainsonine treatment significantly increased the rate of apoptosis.Meanwhile,swainsonine significantly increased the expression levels of LC3-II,ATG5 and Beclin-1 in a time-dose-dependent manner.Transmission electron microscopy revealed that swainsonine treatment increased the accumulation of autophagosomes in the cytoplasm.Furthermore,pretreatment with the autophagy agonist Rapa,the late autophagy inhibitor Baf A1 and the apoptosis inhibitor z-VAD-fmk did not significantly improve swainsonine-induced cytoplasmic vacuolation.ER-Tracker Red fluorescent probe and transmission electron microscope observed that vacuoles appeared around the endoplasmic reticulum,and with the increase of swainsonine dose,the expression of endoplasmic reticulum stress-related proteins Bip,Chop and other proteins increased.Moreover,ubiquitinated proteins gradually accumulated in swainsonine-treated cells,and the expression of cell-like apoptosis inhibitor Alix was down-regulated.Pretreatment with 4-PBA,an endoplasmic reticulum stress inhibitor,could significantly inhibit swainsonine-induced cytoplasmic vacuolated.The above results indicate that swainsonine can induce apoptosis,autophagy and paraptosis of primary renal tubular epithelial cells in rats,and swainsonine-induced cytoplasmic vacuolization and endoplasmic reticulum stress-dependent related to apoptosis.2.Analysis and identification of differential protein expression changes in cells treated with swainsonine based on proteomic analysis.After proteomic analysis,7014 proteins were identified,of which 5697 were quantified.After filtering with thresholds of fold change >1.2 or <0.67 and p <0.05,and finally 95 proteins were found to be up-regulated and64 proteins were down-regulated.GO function enrichment analysis was performed on the differential proteins.Among the 20 cell component classifications with significant impact,4pathways were related to the autophagy-lysosome pathway,and among the 20 more significant molecular function classifications,5 pathways were associated with glycosylation modifications.After verification,it was found that the expression of p62 was increased,while the expression of m-CTSD was decreased,indicating that swainsonine damages lysosome function.Moreover,inhibition of autophagy degradation further promotes swainsonine-induced apoptosis and endoplasmic reticulum stress.The above results indicate that the mechanism of swainsonine poisoning was related to the degradation barrier of autophagy-lysosomal pathway,and also involved the process of glycosylation modification.3.Swainsonine changed the O-GlcNAcylation in primary renal tubular epithelial cells of rats.Western blot detection showed that low dose swainsonine increased the expression of O-GlcNAc modified protein,while high dose swainsonine decreased the expression of O-GlcNAc modified protein.In addition,the expression of OGT and OGA did not change significantly,but the expression of GFAT1,the key enzyme in hexosamine biosynthesis pathway,decreased.Using O-GlcNAcylation agonist TMG and inhibitor Don respectively,it was found that increased O-GlcNAcylation could inhibit apoptosis,promote autophagy and reduce endoplasmic reticulum stress,while decreased O-GlcNAcylation could promote apoptosis,inhibit autophagy and increase endoplasmic reticulum stress,indicating that swainsonine affects autophagy by changing O-GlcNAcylation.According to the results of proteome sequencing,IP assay with O-GlcNAc antibody showed that lysosomal cathepsin CTSD could be modificated by O-GlcNAc,and high dose swainsonine decreased the O-GlcNAcylation level of CTSD protein.Mass spectrometry analysis showed that the 255 s and 260 T of CTSD protein were modified by O-GlcNAc.Furthermore,after protein sequence alignment,it was found that the CTSD protein sequences of rats,humans and mice were highly consistent at position 251-270,indicating that O-GlcNAcylation plays an important role in the function of CTSD protein.To sum up,swainsonine can induce apoptosis,autophagy and paraptosis in primary rat renal tubular epithelial cells,and the cytoplasmic vacuolization induced by swainsonine is related to endoplasmic reticulum stress-dependent paraptosis.Although swainsonine can activate autophagy,it also damages the function of lysosomes,thus inhibiting autophagy degradation and promoting cell death.The specific molecular mechanism is that swainsonine can change the O-GlcNAcylation of CTSD,and then affect the autophagy-lysosome pathway,resulting in the accumulation of wrong proteins and cell death.This study provides a new molecular basis for the mechanism of swainsonine poisoning and a new theoretical basis for the treatment of swainsonine poisoning and resource utilization of locoweed. |
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