Study On The Mechanism Of Action Of Liver Damage Induced By Highly Toxic Forms Of Fluorine In Antarctic Krill

Antarctic krill is the largest single fishable organism in the ocean,and its potential fishery resource value has been valued by all countries in the world.Antarctic krill is a nutrient-rich and important new Marine protein resource,but its high fluoride content limits the safe utilization of high-quality protein.At present,the research on fluoride in Antarctic krill mainly focuses on total fluoride,and there are few reports on the chronic toxicity and cytotoxicity of various forms of fluoride.At the same time,a number of studies have confirmed that fluoride intake has toxic effects on the liver,and there is a lack of research on the bioavailability and related toxicity of fluoride in Antarctic krill.This study aims to screen the fluoride with serious liver damage based on the chronic liver injury effect of various forms of fluoride on rats.To elucidate the mechanism of hepatocyte injury induced by fluoride exposure.The main research contents and results are as follows:Firstly,all forms of fluoride in Krill could damage the liver,and the water-soluble fluoride had the highest degree of damage.According to the stepwise extraction method,fluoride in different forms,including water-soluble fluorine,exchangeable fluorine,oxidized fluorine,organically bound fluorine and residual fluorine,was extracted from Kril Antarctic.The 28-day toxicological experiment in rats was carried out.The results of liver histopathology showed that all forms of fluoride were accumulated in the liver of rats,and the most significant content of water-soluble fluoride was 4.7 mg/kg.Secondly,water soluble fluoride significantly affected the proliferation activity and cell cycle of LX-2 cells.Cell counting kit-8(CCK-8)assay showed that the proliferation activity of LX-2 cells was significantly decreased when the concentration of water soluble fluoride was 25 mg/m L,50 mg/m L and 100 mg/m L,compared with the control group.The cell cycle results showed that water soluble fluoride could significantly increase the proportion of LX-2 cells in S phase,indicating that water soluble fluoride could interfere with signal transduction in the cell cycle,arrest cells in S phase and inhibit cell proliferation.Finally,the results of flow cytometry showed that water soluble fluoride could induce apoptosis of LX-2 cells,and the apoptosis mechanism of LX-2 cells induced by water soluble fluoride was studied from the endogenous and exogenous pathways of apoptosis.Caspase-3 and Annexin V assay showed that the proportion of caspase-3 in LX-2 cells treated with water soluble fluoride was higher than that in the control group,and the apoptosis rate was also higher than that in the control group.The main pathways affecting apoptosis are the endogenous pathway and the death receptor pathway.Excessive ROS production is the main cause of oxidative stress.2,7-dichlorofluorescein diacetate(DCFH-DA)fluorescence staining showed that the fluorescence intensity of intracellular ROS increased in LX-2 cells treated with water soluble fluoride in a dose-dependent manner.Excessive ROS also led to the production of Malondialdehyde(MDA),a lipid peroxidation product.Compared with the control group,the content of MDA after water soluble fluoride treatment was significantly increased.Secondly,the expression of intracellular Superoxide Dismutase(SOD)and the related antioxidant glutathione(r-glutamyl cysteingl +glycine,GSH)was also affected by oxidative stress.The results showed that compared with the control group,the activity of SOD and the content of GSH in the water-soluble fluoride treated group decreased significantly.Western blot was used to detect the expression levels of endogenous apoptosis-related proteins cleaved-caspase-3,Bax,Bcl-2 and caspase-9.The endogenous apoptosis-related proteins Bax,cleaved-caspase-3 and caspase-9 were significantly up-regulated,while Bcl-2 was significantly down-regulated.Exogenous pathway: By detecting the expression levels of death receptor pathway related proteins,it was found that caspase-8 and tumor necrosis factor receptor(Fas)were significantly up-regulated.These results suggested that water soluble fluoride induced apoptosis by inhibiting the expression of Bcl-2 and promoting the expression of Bax,and induced the increase of membrane permeability and activation of caspase-9 and caspase-3.Water-soluble fluoride stimulates Fas through the death receptor pathway,then activates caspase-8 and caspase-3 to induce apoptosis.In conclusion,water-soluble fluoride from Krill induces apoptosis of LX-2hepatocytes through oxidative stress and death receptor pathways.