Study On The Protective Effect And Mechanism Of Ganoderma Lucidum Polysaccharides On Intestinal Cell Damage Induced By Acrylamide

In the environment,acrylamide(AA) is a potentially toxic pollutant,also it is produced during the thermal processing of food.Some research shows that AA can cause damage to the structure and function of small intestinal mucosa and intestinal epithelial cells.And oxidative stress and apoptosis induced by ROS are the potential mechanisms of AA's damaging to small intestinal cells.Therefore,from the mechanism,looking for natural products which can inhibit the generation of ROS is a promising approach that can prevent and inhibit the damage of intestine which is caused by AA.Ganoderma lucidum polysaccharide is the mainly active ingredient of ganoderma atrum.Ganoderma lucidum polysaccharide is the main active ingredient of Ganoderma lucidum.Preliminary studies have proved that ganoderma lucidum polysaccharide has multiple biological activities like immune regulation,anti-aging,anti-tumor,anti-diabetic,myocardial protection and so on.Ganoderma lucidum polysaccharide playing a variety of biological activities is the central link and common mechanism of inhibiting of ROS generation.Therefore,this subject mainly discusses the protective effect and possible protective mechanism of Ganoderma lucidum polysaccharide from the aspect of AA toxicity to small intestine cells.It has important theoretical significance and practical value for the treatment or prevention of AA toxicity,and also can provide scientific guidance and experimental basis for dietary nutrition.The main contents of this study are as follows:1.First of all,In vitro experiment,in order to investigate the protective role of Ganoderma lucidum polysaccharide(PSG-F₂) on AA induced oxidative damage in IEC-6 cells was investigated by measuring the cell viability and intracellular antioxidant enzyme activity.The main results are as follows:(1)In this study,5mmol/L AA was selected as the best induction concentration,and the cell survival rate was as high as 46%.(2)Compared with the blank control group,1-10 g/ml in the range of PSG-F₂ can cause the reduction of cell viability,but there is no significant difference.Thus,different mass concentrations(20?40?80?160?g/ml)of PSG-F₂ were selected for further study.(3)Compared with the control group,cell viability decreased significantly after AA being processed at different times.Ganoderma lucidum polysaccharide and NAC can relieve low survival rate which was caused by AA,can significantly reduce the leakage of LDH in cell culture medium,can increase SOD and GSH-Px activity in cellar,and can reduce the content of MDA.Conclusion:Ganoderma lucidum polysaccharides can protect AA induced IEC-6 cells from oxidative damage by increasing the activity of antioxidant enzymes and inhibiting lipid peroxidation in IEC-6 cells.2.By using flow cytometry and immunoblotting,we studied different concentrations of PSG-F₂(20,40,80 and 160?g·ml^-1) to the protective effect of AA on IEC-6 cell apoptosis and its potential mechanism.The main results are as follows:(1)Compared with the normal group,the ROS in the AA group increased significantly.PSG-F₂ can reduce the amount of intracellular ROS production induced by AA to the normal level,and compared with AA treatment group,mitochondrial membrane potential can be reduced by about 20.5%-24.9%.(2)AA can induce apoptosis of IEC-6 cells,compared with AA treatment group,the apoptosis rate of PSG-F₂ group was decreased by 10.1%-13.7%.(3)Compared with the blank control group,AA can induce the expression of cytochrome c and caspase-3 protein in the cell,and accompanied by activation of Cleaved caspase-9 and Cleaved caspase-3,having significant difference(P<0.01).This phenomenon shows that after being stimulated by AA,cytochrome c is released into the cytoplasm to activate the endogenous mitochondrial apoptotic pathway.PSG-F₂ or NAC also prevented the AA-induced increases of the protein expression of caspase 3,Cleaved caspase-9,Cleaved caspase-3 and cytochrome c.Especially in the 20?40?160 ug/ml PSG-F₂group,the protective effect was the most obvious,and better than the NAC group.The results show that ganoderma lucidum polysaccharide can inhibit the apoptosis of AA cells by regulating the expression of related proteins in the endogenous mitochondrial apoptosis pathway.(4)Compared with group AA,the expression levels of p38 and p-p38 in the cells were increased after adding different concentrations of PSG-F₂.For the expression of p38 protein,the effect of PSG-F₂ group was significantly better than that of NAC group.Especially in the 20 ug/ml PSG-F₂ group,the protective effect was the most obvious.For the expression of p-p38 protein,especially in the160 ug/ml PSG-F₂group,the protective effect was the most obvious,and better than the NAC group.On the expression of ERK protein,there was no significant change between the treatment groups(P>0.05).For the expression of p-ERK protein,the high concentration of PSG-F₂(160 ug/ml)can significantly increase the expression of p-ERK(P<0.01),and the protective effect is better than NAC.3.Through the establishment of mice in vivo,we can study the protective effect of Ganoderma lucidum polysaccharides on intestinal injury induced by AA in rats.The results show that the activity of antioxidant enzyme system,such as SOD,GSH-Px,CAT,T-GSH,GSH increased significantly,and the content of MDA decreased significantly compared with the AA group.In additon,compared with AA group,small intestine histopathology showed that the intestinal villous epithelial cells were damaged significantly alleviate by Ganoderma lucidum polysaccharides.Meanwhile,different concentrations of Ganoderma lucidum polysaccharides can significantly reduce the level of uric acid and alkaline phosphatase in rat intestine,down the regulation of proinflammatory cytokines IL-1??IL-2?TNF-a,significantly increase the contents of anti-inflammatory factor IL-4 and IL-10,obviously decrease the content of intestinal mucosal permeability index:D-lactate and endothelin-1.The experimental results show that Ganoderma lucidum polysaccharides can maintain the redox balance of system,reduce intestinal inflammation,improve intestinal mucosal immune function,reduce intestinal permeability and inhibit oxidative damage on the body caused by acrylamide.The research shows that Ganoderma lucidum polysaccharide as the edible components of a natural source can prevent the toxicity of AA,can play an important role in protecting AA from oxidative damage in small intestine,and can provide a theoretical basis for further development of Ganoderma lucidum polysaccharide.