TRPML1-mediated Autophagy As A Repairment Target Of Aflatoxin B1-induced Porcine Intestinal Epithelial Cells

The purpose of the present study was to investigate the efficacy of TRPML1 as a target in the prevention and treatment of intestinal injury caused by Aflatoxin B1（AFB1）in pigs.The experiment took porcine intestinal epithelial cells（IPEC-J2）as the research object.The AFB1 exposure model was established and ROS,Ca²⁺concentration,apoptosis,autophagy,and autophagy flux were detected by flow cytometry,Western blot,immunofluorescence,and transmission electron microscopy.Then,reactive oxygen species（ROS）scavengers（N-acetylcysteine,NAC）,autophagy inhibitors（3-methyladenine,3-MA;Chloroquine CQ）,and lysosomal membrane cation channel protein inhibitor TRPML1（ML-SI1）were used to investigate the mechanism of ROS,autophagy,and TRPML1 in AFB1 exposure model.The results are as follows:1.IPEC-J2 cells were exposed to different concentrations of AFB1（0,2,5,10,20,50,100μg/m L）for 24 h,and the cell viability decreased to 80%and 60%in a dose-dependent manner when AFB1 concentration was 5μg/m L and 10μg/m L.2.Compared with 0μg/m L AFB1 treatment,5μg/m L and 10μg/m L AFB1 treatment significantly upregulated ROS levels and apoptosis rate.Apoptotic cells were enhanced remarkably by AO/EB staining.The expressions of Caspase3,Caspase 9,Bax were ameliorated,while expression of the Bcl-2 gene was declined.3.Compared with 0μg/m L AFB1 treatment,5μg/m L and 10μg/m L AFB1 treatment showed that mitochondria were vacuolated,ridges were blurred,and the number of autophagosomes was increased.MDC staining and LC3 staining verified that the number of acidic autophagic vesicles and LC3 fluorescence puncta were increased dramatically.The mRNA and/or protein expression of autophagy-related genes such as LC3 II and SQSTM1were increased.4.Compared with 0μg/m L AFB1 treatment,5μg/m L and 10μg/m L AFB1 treatment showed that the expression of TRPML1 was significantly increased.The result of the flow cytometry revealed that the level of cellular Ca²⁺was significantly increased.Confocal laser microscopy results indicated that the co-localization signals of autophagosome marker protein LC3 and lysosomal membrane protein LAMP-1 were weakened.After the infection of RFP-GFP-LC3 lentivirus,the intracellular yellow fluorescence signal（GFP and RFP co-location）was increased significantly,indicating the autophagy flux was blocked.5.After co-treatment with 100μM NAC and 10μg/m L AFB1,compared with 10μg/m L AFB1 treatment,the level of cellular ROS and the rate of apoptosis were significantly reduced.The protein level of Cleaved-Caspase3/Caspase3 was decreased,and the protein levels of autophagy-related proteins（STSQM1,LC3 II/LC3 I,and TRPML1）were reduced.6.After co-treatment with 500μM 3-MA and 10μg/m L AFB1,compared with 10μg/m L AFB1 treatment,the level of cellular ROS and the rate of apoptosis were decreased.The co-localization signal of LC3 and LAMP-1 was enhanced.The intracellular yellow fluorescent signal was downregulated.The protein level of Cleaved-Caspase3/Caspase3 was decreased,and the protein levels of autophagy-related proteins（STSQM1,LC3 II/LC3 I,and TRPML1）were reduced.After co-treatment with 50μM CQ and 10μg/m L AFB1,compared with 10μg/m L AFB1 treatment,the level of cellular ROS and the rate of apoptosis were increased.The co-localization signal of LC3 and LAMP-1 was significantly mitigated.The intracellular yellow fluorescence signal was significantly upregulated.The protein level of Cleaved-Caspase3/Caspase3 was augmented,and the protein levels of autophagy-related proteins（STSQM1,LC3 II/LC3 I,and TRPML1）were increased.7.After co-treatment with 1.25μM ML-SI1 and 10μg/m L AFB1,compared with 10μg/m L AFB1 treatment,the levels of cellular ROS,apoptosis rate,and the level of intracellular Ca²⁺were significantly declined.The co-localization signal of LC3 and LAMP-1 was significantly ameliorated.The intracellular yellow fluorescence signal was remarkably decreased.The protein level of Cleaved-Caspase3/Caspase3 was decreased,and the protein levels of autophagy-related proteins（STSQM1,LC3 II/LC3 I,and TRPML1）were reduced.Collectively,AFB1 activated ROS/TRPML1 pathway which blocking the autophagic flux,leading to IPEC-J2 cell apoptosis.