The Protective Mechanism Of Ganoderma Atrum Polysaccharide On IEC-6 Cell Injury Induced By Acrylamide

Acrylamide is produced by the Maillard reaction of reducing sugars with asparagine during food cooking,such as frying,baking,or grilling.According to previous studies,food containing AA can cause homeostasis disorders,genetic toxicity,neurotoxicity or reproductive disorders.Studies on various animals have shown that acrylamide seems to be rapidly distributed to tissues,mainly muscle tissue,gastrointestinal tract,lungs,skin and bone marrow.In the human body,acrylamide can be absorbed highly and quickly in the intestine.Therefore,it is necessary to understand the transport mechanism of acrylamide in intestinal tract and its regulation of intestinal epithelial cell transformation pathway.Ganoderma atrum polysaccharide（PSG-1）is the main bioactive component of ganoderma artum.PSG-1 and its main acidic component,PSG-1-F₂（PSG-F₂）,have good antioxidant,immunomodulatory,anti-cancer,anti-aging,cardiovascular protection and anti-diabetes activities in vivo and in vitro.Our previous in vitro and in vivo experiments found that polysaccharides from Ganoderma atrum have antioxidant ability on the oxidative damage of intestinal tissue induced by acrylamide.However,the protective mechanism of polysaccharides from Ganoderma atrum on the cell damage induced by acrylamide remains unclear and needs further study.Therefore,in this study,RNA-seq technology was used to explore the protective effect of polysaccharides from Ganoderma melanoderma on cell damage caused by acrylamide.The main research results are as follows1、Based on RNA-seq analysis of PSG-F₂on the AA-induced IEC-6 cell damage gene expression and signal pathways.Compared with the control group,a total of 1559 DEGs were identified in the AA group,of which 621 DEGs were up-regulated and 938 DEGs were down-regulated;in the 320μg/m L PSG-F₂group,a total of 1312 DEGs were identified,540 DEGs were up-regulated,and 772 DEGs were down-regulated.Compared with the AA group,a total of 438 DEGs were identified in the 320μg/m L PSG-F₂group,of which 133 DEGs were up-regulated and 305 DEGs were down-regulated.GO and KEGG enrichment analysis were used for the acquired transcriptome information.The data showed that autophagy,apoptosis and ferroptosis gene sets were significantly up-regulated after AA treatment,suggesting that AA may have a toxic effect on cells by regulating autophagy and ferroptosis.Among them,related signal pathways include MAPK signal pathway、cell cycle、Hippo signal pathway、etc.,while PSG-F₂pretreatment can slow down this damage through the cell cycle、MAPK signal pathway、lysosome and PI3K-Akt signal pathway.2、Experimental validation of the role of MAPK signaling pathway,and the results showed that:Compared with the control group,IEC-6 cells reached half mortality after treated with AA for 8h,cell shrinkage and adhesion became worse,and the levels of SOD、GSH、mitochondrial membrane potential MMP、Cytochromatic c、intestinal tight junction proteins ZO-1、Occludin and Claudin 1decreased.Meanwhile,the increased levels of peroxide products MDA、mitochondrial mt ROS、p-p38、p-JNK、p-ERK and other proteins in cells indicate that AA can cause oxidative damage through the activation of MAPK pathway,leading to barrier damage.Compared with AA group,PSG-F₂reduced cell morphological damage,improved cell adhesion,enhanced the activities of antioxidant enzyme system SOD and GSH,and increased the protein levels of membrane potential MMP、Cytopigment c、ZO-1、Occludin and Claudin 1.The levels of peroxide products MDA、mitochondrial mt ROS、p-p38、p-JNK、p-ERK and other proteins were decreased.These results indicated that PSG-F₂pretreatment could inhibit oxidative damage of cells and intestinal barrier damage caused by AA by inhibiting MAPK pathway.Based on RNA-seq data,further explore the molecular mechanism of PSG-F₂inhibiting AA-induced autophagy and ferroptosis in IEC-6cells.Experimental results show that PSG-F₂reduces the expression of LC3 and Beclin 1 through the PI3K-AKT-m TOR signaling pathway,maintained iron homeostasis and controlled ferroptosis through inhibiting lipid ROS production and Nrf2-SLC7A11-HO-1 signaling pathway.3、Validation of molecular mechanisms of autophagy and ferroptosis.The experimental results showed that compared with the control group,AA increased the Fe concentration、lipid ROS level、LC3、Beclin 1、PI3K、Akt、m TOR、Nrf2、SLC7A11、HO-1、COX-2 protein levels in IEC-6 cells.By reducing the expression of GPX4 protein and adopting the mitochondrial inhibitor TEMPO、mitochondrial ROS、MMP and cytochrome c could be reduced,while cell autophagy proteins LC3、Beclin 1 and cell ferroptosis protein COX-2 could be decreased,and the level of GPX4 could be increased.These results indicate that inhibition of mitochondrial damage can inhibit autophagy and ferroptosis.Lysosomal inhibitor CQ can reduce autophagy protein LC3、Beclin 1 and cell ferroptosis protein COX-2,increase the level of GPX4,and reduce Fe concentration,suggesting that inhibition of autophagy can inhibit ferroptosis.The above results indicate that AA produces mitochondrial ROS through mitochondrial damage,leading to the decrease of mitochondrial membrane potential and the release of Cytochrome c,which leads to the accumulation of lipid ROS,resulting in the imbalance of cellular REDOX and excess autophagy,resulting in the increase of cellular free iron content,activation of Nrf2-SLC7A11-HO-1 pathway and finally ferroptosis.Compared with AA group,PSG-F₂decreased Fe concentration、lipid ROS level、LC3、Beclin 1、PI3K、Akt、m TOR、Nrf2、SLC7A11、HO-1、COX-2 protein levels in cells,and increased GPX4 expression.These results indicate that PSG-F₂may inhibit autophagy and Nrf2-SLC7A11-HO-1 pathway by slowing mitochondrial damage and activating PI3K-Akt-m TOR signaling pathway to maintain cell iron homeostasis and control cell ferroptosis.