Effect And Mechanism Of Oxidative Stress Induced By Indene At Cellular And Molecular Level In Coelomocytes Of Earthworm

In the process of petroleum exploitation,transportation,refining and chemical production and utilization,due to improper operation or storage management and other problems will cause the leakage of petroleum hydrocarbons,resulting in environmental pollution.The composition of petroleum hydrocarbons is complex,mainly including alkanes,cycloalkanes and aromatic hydrocarbons.Indene(IND)is a thick aromatic hydrocarbon and an important part of petroleum hydrocarbons.IND accounts for 5%-15%of petroleum hydrocarbon C9 fractions.IND is easy to enter the soil in the process of utilization,which has a certain negative impact on the soil environment,resulting in soil ecological environment risks,and then harm the respiratory system,liver,kidney and other organs of the human body.However,in the field of scientific research,the toxic effect and mechanism of IND are still insufficient,especially the oxidative stress effect on soil typical organisms at cellular and molecular level and its corresponding mechanism have not been elucidated,which further affects the formulation of prevention and control strategies for indene petroleum hydrocarbon pollutants.In this study,the coelomocytes and antioxidant enzymes from the Eisenia foetida were selected as the research subjects at the cellular and molecular levels,and the effects and mechanisms of IND induced oxidative stress were studied.In addition,the interaction model of IND with direct antioxidant enzyme superoxide dismutase(SOD)and indirect antioxidant enzyme lysozyme(LYZ)was established to explore the interaction mechanism.This paper is mainly divided into the following four aspects::In chapter 1,the harm of petroleum hydrocarbon pollution and the research progress of IND toxicity,and describes the composition of antioxidant system,the concept and harm of oxidative stress,research methods,content and significance of this study were described.In chapter 2,the oxidative damage and primary antioxidant system damage caused by IND to coelomocytes of earthworm were studied.The coelomocytes of earthworm were selected as experimental objects to explore the effect and mechanism of oxidative stress induced by IND on coelomocytes of earthworm.The effects of IND on cell viability,reactive oxygen species(ROS)level,total antioxidant capacity(T-AOC),SOD,POD,CAT,LYZ activity,content of reduced glutathione(GSH),cell membrane damage and carboxylesterase(CarE)activity of earthworm were studied.The experimental results showed that the intracellular ROS level increased significantly to 269.6%of the control group after 24 h exposure to IND,and the redox equilibrium was broken,resulting in oxidative stress effect.The activities of SOD,POD,CAT and LYZ and other enzymes antioxidants were affected by ROS to varying degrees.The non-enzymatic antioxidant GSH were also continuously depleted.Then the T-AOC of cells changed.The activity of detoxification enzyme CarE was increased in a certain concentration range.At the same time,IND also changed the permeability and fluidity of cell membrane,leading to lipid peroxidation of cell membrane,resulting in decreased cell vitality in body cavity.In chapter 3,the mechanism of action of IND on direct antioxidant enzyme SOD and indirect antioxidant enzyme LYZ was studied at molecular level.The effects of IND exposure on SOD and LYZ enzyme activities were detected by using SOD and LYZ activity detection kits.The effects of IND on the molecular structure and conformation of SOD and LYZ were studied by multispectral technique.Thermodynamic parameters such as binding constant,binding site,enthalpy change and entropy change were calculated by isothermal calorimetric titration experiment(ITC).The binding of IND with SOD and LYZ was further simulated by molecular docking technology,and the interaction model was established.The results showed that IND combined with SOD and LYZ to form aggregates with particle sizes larger than the protein itself.The binding forces were hydrophobic,and the binding constants were 4.95×103 M-1 and 1.45×103 M-1,respectively.IND exposure enhanced the endogenous fluorescence of SOD and LYZ and affected the microenvironment of the tyrosine residue of SOD and the tryptophan residue of LYZ,reducing their hydrophilicity.IND loosened the skeleton structure of SOD and LYZ,resulting in disfolding phenomenon,and changed the contents of a helix andβ folding secondary structure in molecular structure.IND mainly binded to the interface of the two subunits of SOD and the concave position on the surface of LYZ molecule.At the same time,the binding pocket of IND contained aspartic acid residue Asp 52,which was crucial to the activity of LYZ.This binding mode increased the contact area between the active center of SOD and LYZ and the substrate,thus improving the activities of SOD and LYZ.In chapter 4,the main conclusions,innovation points,shortcomings of this paper and the future research prospects of this field were summarized and analyzed.In the next step,it is necessary to further study the correlation between the oxidative stress effect generated by cells exposed to IND and related signaling pathways,and clarify the mechanism of oxidative stress effect induced by IND from the perspective of signaling pathways.