The Regulatory Mechanism Of MTOR-related Signaling Pathway In Swainsonine-induced Autophagy Of HT22 Cells

Locoweed is a general term for poisonous plants belonging to the genus Oxytropis and Astragalus of the family Leguminosae.It is the main poisonous weed that endangers the sustainable development of grassland animal husbandry worldwide.Our country has a natural grassland area of 400 million hm²,and the poisonous weed damage area is about 33.3 million hm²,of which the poisonous locust weed is the most serious,accounting for about 33%of the poisonous weed damage area.Its main toxic component is indolizidine alkaloid-swainsonine（SW）.The chemical structure of SW is highly similar to that of mannose in animals,and has a very high affinity for mannosidase.It can competitively inhibit the activity of class Ⅱα-mannosidase,leading to disorder of glucose metabolism,and then the locoism characterized by extensive vacuolar degeneration of histiocytes occurs.Autophagy is a metabolic pathway for self-protection of eukaryotic cells.It wraps damaged organelles,macromolecules,etc.with part of the cytoplasm to form autophagosomes,which are then combined with lysosomes to form autophagolysosomes,which are responsible for the encapsulated contents.It degrades and recycles,realizes the renewal of substances and organelles,and maintains the balance of intracellular substances and energy supply.Studies have shown that when autophagy is impaired,cells exhibit vacuolar-like lesions.However,the role and mechanism of autophagy in SW-induced vacuolar degeneration is unclear.In view of this,this study used Mouse Hippocampal Neurons cell line（HT22）as the experimental cell model to carry out related research and obtained the following research results:1.Swainsonine causes HT22 cell damage.After SW treatment,the vacuolar degeneration in HT22 cells was observed by inverted phase contrast microscope,and it was found that the vacuolar degeneration gradually increased with the prolongation of treatment dose and treatment time;qRT-PCR was used to detect Golgiα-mannosidase Ⅱ and lysosomalα.-The expression of mannosidase changed,and it was found that their expression was significantly down-regulated with the prolongation of treatment dose and treatment time.Bafilomycin A1 can improve the above two conditions and slow down the toxicity of SW.2.Swainsonine induces autophagy in HT22 cells.Expression of autophagy marker LC3 protein and gene.When cells were treated with different concentrations of SW for 12 h,LC3-Ⅱ/β-actin increased in a dose-dependent manner,and the mRNA expression also had the same change,and the difference between groups was statistically significant.Subsequently,cells were treated with 0.8 mg/mL SW for different times,and LC3-Ⅱ/β-actin also increased in a time-dependent manner,with the same mRNA expression changes,and the differences between each time point and the control group were statistically significant.Fluorescent staining.When cells were treated with different concentrations of SW for 12h,the MDC staining in the control group was diffuse,while the cells in the treatment group showed fluorescent clusters,indicating extensive MDC-positive autophagic vacuoles;The results of LC3-Ⅱ fluorescence staining showed that SW increased the formation of LC3-Ⅱ fluorescent foci in HT22 cells,and the fluorescence intensity increased with the increase of dose.MDC-positive autophagic vacuoles and LC3-Ⅱ puncta were significantly increased after treatment with 0.8 mg/mL SW for different times.Observation by transmission electron microscopy showed that the number of autophagic vesicles increased significantly with the change of SW concentration and treatment time.The above results indicate that SW can induce a large number of autophagosomes with obvious dose and time effects.3.Swainsonine activates autophagy and leads to blocked autophagic degradation.The test found that ATG5,Beclin1 and p62 protein and mRNA expression levels increased with the increase of dose and time.Elevation of ATG5 and Beclin1,showing increased autophagosomes,indicates elevated levels of intracellular autophagy,and accumulation of p62 indicates that the final degradation of autophagy is blocked.There are two possibilities for the blocked autophagic degradation,one is that the function of lysosomes is damaged,and the other is that the fusion of autophagosomes and lysosomes is hindered.Therefore,the changes of lysosomal membrane-associated protein 1（Lamp1）in SW treated with different concentrations and time were detected by Western-blot and qRT-PCR,showing that the changes of Lamp1 protein and mRNA expression increased with obvious time and time dose effect;Subsequently,lysosomes were stained with Lyso Sensor^?Green DND-189 and Lyso-Tracker Red,and it was found that with increasing challenge dose,the number of lysosomes increased,but the pH of the cavity increased.It is suggested that the blocked intracellular autophagic degradation after SW treatment is related to the impaired lysosomal function.4.mTOR-related pathways are involved in the regulation of swainsonine-induced autophagy in HT22 cells.Western-blot was used to detect the expression of autophagy signal mTOR pathway-related proteins.HT22 cells were treated with different concentrations of SW,with the increase of SW concentration,the protein expression levels of p-PI3K,p-AKT,p-ERK,p-AMPK,p-mTOR,p-p70S6K and p-4EBP1 decreased significantly,and the total PI3K,AKT,ERK,AMPK,mTOR,p70S6K and 4EBP1 remained unchanged,and p53 protein expression levels were significantly increased.The results showed that the PI3K/AKT/mTOR,ERK/mTOR and p53/mTOR autophagy signaling pathways were involved in the regulation of SW-induced autophagy in HT22 cells,while the AMPK/mTOR pathway was not involved in this regulation.The above results indicate that SW induces autophagy in HT22 cells by impairing the lysosomal function of HT22 cells,resulting in the inability of autophagosomes to be completely degraded.It was further found that PI3K/AKT/mTOR and ERK/mTOR signaling pathways play a positive regulatory role in SW-induced autophagy in HT22 cells.The p53/mTOR signaling pathway plays a negative regulatory role,while the AMPK/mTOR signaling pathway does not participate in this regulatory process.The results of this experiment can further reveal the mechanism of SW toxicity and provide an important theoretical basis for the prevention and treatment of locust poisoning.