Characterization Of Pumpkin Polysaccharide And Its Intervention Effect On BRL Cytotoxicity Induced By Carbendazim

Carbendazim is a widely used broad-spectrum fungicide with high residual rate and concentration and it had strong multi-system toxicity to the organism.Thus,its potential health risks have been concerned.Existing studies have shown that diabetic patients,susceptible groups,are more sensitive to toxicity of pollutants.However,their susceptibility to toxicity caused by CBZ has not been reported,which is in urgent need of study.Studies have found that pumpkin polysaccharide has excellent hypoglycemic and hypolipidemic effects,but whether it could alleviate the toxic effects of CBZ exposure on diabetic population and effectively reduce the risk of CBZ exposure in diabetic population remains to be studied.Therefore,the structure of salt-washed pumpkin polysaccharide fraction(ZPP2)was firstly characterized in this paper.The cell exposure model was established for investigation of the toxic effects of CBZ on normal BRL cells and IR-BRL cells,analyzation of the susceptibility to toxicity and revelation the susceptibility mechanism.On above basis,the nutritional intervention effect of ZPP2 on BRL cytotoxicity induced by CBZ exposure was further discussed,and the intervention mechanism was clarified.This study can provide a basis for prevention and treatment of toxicity induced by CBZ exposure to diabetic patients through dietary nutrition intervention,which is of great significance for the health of diabetic population.The main research contents are as follows:(1)The structure of ZPP2 was characterized by IR,methylation,GC-MS and NMR.Results indicate that the ZPP2 was a kind of heteropolysaccharide composed by Man,Rha,Glu A,Glu,Gal,Xyl and Ara with the mole ratio of 2.0:1.2:1.0:12.6:4.4:3.8:1.2.The main chain of ZPP2 was mainly composed of T-linked-D-Glup,1,6-linked-Galp,1,4-linked-Glup and 1,6-linked-Glup with the molar ratio of 9.2:12.7:11.1:17.4.ZPP2 was a kind of galactoglucomannan.(2)The insulin resistance BRL cell model was established to study the toxic effect of CBZ exposure on BRL cells,then analyze the susceptibility of cells in different states to CBZ toxicity and further discuss the susceptibility mechanism.The results showed that CBZ could inhibit glucose uptake and utilization by cells and increase intracellular lipid levels by promoting intracellular lipid synthesis and the expression of gluconeogenic genes and inhibiting the expression of lipid metabolism-related genes,thereby cause the disorder of glucose and lipid metabolism.CBZ exposure inhibited the activity of antioxidant enzymes and promotes the secretion of inflammatory factors by up-regulating the expression of genes related to cellular inflammation,oxidative stress and apoptosis,resulting in the accumulation of intracellular ROS and MDA,finally inducing inflammatory and oxidative damage to cells.It was found that CBZ induced endoplasmic reticulum stress and apoptosis by activating PERK/e IF-2α and Bcl-2/Bax/Caspase-3 pathways,interfered with cell metabolism and caused endocrine and metabolic toxicity.Susceptibility analysis showed that there was a significant interaction between the establishment of insulin resistance model and the exposure concentration of CBZ,indicating that IR-BRL cells were more susceptible to cytotoxicity caused by CBZ.(3)Normal BRL and IR-BRL cells were used as research objects to further explore the nutritional intervention effect of ZPP2 on the cytotoxicity of IR-BRL induced by CBZ exposure,and reveal its intervention mechanism.The results indicated that ZPP2 could ameliorate the disorder of glucose and lipid metabolism caused by CBZ exposure by inhibiting the abnormal increase of intracellular lipid level and promoting glucose uptake and utilization.It also could repair the oxidative damage and inflammatory damage of cells by inhibiting the oxidative stress state and inflammatory response of cells,evidenced by increase of the activity of SOD enzyme,decrease of the activity of Caspase-3 enzyme,inhibition of the accumulation of ROS and MDA in cells and the secretion of inflammatory factors.The effect of pre-intervention was significantly better than simultaneous intervention and post-intervention,which is the best way of intervention.The results of the intervention mechanism study showed that ZPP2 could regulate the expression of lipid synthesis,gluconeogenesis,oxidative stress,cellular inflammation and apoptosis genes by inhibiting the activation of PERK endoplasmic reticulum stress pathway,thereby promoting the absorption and transport of glucose and inhibiting the synthesis of cholesterol and fatty acids and promoting their metabolism.In addition,ZPP2 also alleviated cell-damage induced inflammatory,oxidative stress and cell apoptosis to realize the protective effect of ZPP2 on IR-BRL cytotoxicity induced by CBZ.