Investigation Of The Toxicological Mechanism Of Beauvericin On Silkworm BmE Cells And Human HEK293T Cells

Beauvericin（BEA）is a Fusarium toxin,a secondary metabolite produced by parasites such as beauveria bassiana during infection of arthropods.Due to its strong cytotoxicity to insect cells,insecticidal properties,and good control effect on a variety of pests such as red-headed lily fly,Aedes aegyptida,grass blind bug,fall armyworm,and wheat bifur aphid,beauvericin has been widely used as a biopesticide.Beauvericin is also one of the active ingredients of traditional Chinese medicine zombie silkworm,which has anti-tumor,bacteriostatic,antiviral,anticonvulsant,anti-tuberculosis,antimalarial parasite and other therapeutic effects,and has great medicinal value,but at the same time shows certain toxic side effects on a variety of cell lines.In addition,beauvericin has also been found to be a contaminant of a variety of cereals and cereal products,and its content is one of the key indicators in the food safety risk assessment of cereals and their products,vegetable oils and nuts.Whether it is used as a biopesticide or a therapeutic drug,the insecticidal properties and cytotoxicological mechanism of beauvericin are not fully understood.The systematic analysis of the toxicological mechanism of beauvericin may provide a positive reference for the development of new and efficient biological pesticides based on beauvericin,the modification of low-toxicity beauvericin lead compounds,and the revision of food safety testing standards.For the study of cytotoxicology,our laboratory developed a new method based on genome-wide CRISPR editing library,and analyzed the toxicological mechanism of two common pollution factors on silkworm cells with environmental pollutants fluorine and cadmium as targets,and verified the effectiveness and efficiency of the method.Therefore,this paper selected beauvericin as the target to study the toxicological mechanism based on genome-wide CRISPR editing library in the natural host of Beauveria bassiana,model organism silkworm BmE cells in Lepidoptera,and human embryonic kidney cells（HEK293T）.In this study,the whole genome editing cell library was used to combine cell biology and molecular biology to screen the target genes of beauvericin in silkworm BmE cells and HEK293T cells,and explore the response mechanism of BmE cells and HEK293T cells to beauvericin.The main results achieved are as follows:1.The whole genome editing library of BmE cells was used to identify the gene responding to beauvericin in silkwormsIn this part of the study,we used the genome-wide editing silkworm BmE cell library constructed in our laboratory to realize the screening of genes related to beauvericin treatment.First,we verified that beauvericin is toxic to silkworm BmE cells and individuals,and beauvericin causes significant changes in cell morphology and even accompanied by cell death at higher concentrations.Detection of the nucleus showed that the nucleus was divided and wrinkled.The activity detection experiment showed that the semi-inhibitory concentration（IC50）of beauvericin was 4.42μM.Subsequently,the silkworm BmE cell library was treated with a medium containing 4μM beauvericin,each group was about 2×10⁷ cells,3 replicates,the same set of control group library cells were cultured with normal medium,after 7 days of continuous culture,the control cells grew normally,proliferated to about 2 times the original,and the number of cells in the experimental group was reduced to about 10%of the number of control cells,the surviving cells were collected,the genome was extracted,and the sg RNA region was amplified by PCR.PCR products are sent to the company for next-generation sequencing.Analysis of sequencing results revealed a total of 548significantly enriched genes and 890 significant consuming genes.Some of the top significantly enriched candidate target genes were verified,and it was found that the knockout of genes Litaf and CYP9E2 significantly increased the tolerance of silkworm BmE cells to beauvericin,proving that the screening results had high confidence,and the 1438 genes identified could be used for subsequent analysis.2.The HEK293T whole genome editing library was used to identify the gene of human cells responding to beauvericinConsidering that beauvericin has great application potential in the treatment of human diseases,but its toxic side effects on cells need to be solved urgently,this part of the content is based on the screening of silkworm BmE cell libraries,using the widely used HEK293T whole genome CRISPR cell editing library,the screening and identification of beauvericin response genes in human cells based on library screening is carried out.First,we determined the toxicity of beauvericin to HEK293T cells,which begin to round and accompany float with increasing time and treatment concentration.DAPI staining showed that the nucleus of the cell was wrinkled and divided into small pieces after beauvericin treatment.Cell viability assays determined that the IC50 of HEK293T cells treated with beauvericin was 6.498μM.Referring to the growth cycle and state of cells during IC50 and library screening,the beauvericin concentration for library screening was finally determined to be 2.5μM.Subsequently,the human HEK293T cell library was treated with a medium containing 2.5μM beauvericin,about2×10⁷ cells per group,3 replicates,the same set of control group library cells were cultured with normal medium,after 7 days of continuous culture,the control cells grew normally,proliferated to about 2 times the original,and the number of cells in the experimental group was reduced to about 10%of the number of control cells,the surviving cells were collected,the genome was extracted,and the sg RNA region was amplified in large numbers by PCR.PCR products are sent to the company for next-generation sequencing.By screening beauvericin through the HEK293T genome-wide knockout cell library,we identified a total of 1074 significantly enriched genes and 1150 significantly consumed genes.We verified the function of a single gene in some of the top genes screened,and found that knocking out the TP53AIP1 gene in cells significantly increased the tolerance of HEK293T to beauvericin,confirming that the screening results also had high confidence,which laid a foundation for further analysis of the toxicological mechanism of beauvericin.3.The connection between the mechanism of action of beauvericin on BmE cells and HEK293T cellsBased on the previous two parts,the key genes in response to beauvericin treatment in BmE and HEK293T cells were fully laid,and this part focuses on the comparative analysis of the two.First,GO function annotation analysis was performed on the identified significantly enriched genes and significant consuming genes,and we found that in both BmE and HEK293T cells,the exposure treatment of white stiffcin may cause cytotoxicity by affecting the function of certain proteins or competing with certain ion(Ca²⁺,Na⁺,K⁺)receptors,disturbing intracellular ion homeostasis.Further subcellular localization results in silkworms showed that the key genes in the mechanism of action of silkworms in response to beauvericin were mainly in the nucleus,indicating that the toxic mechanism caused by beauvericin was mainly intracellular reaction.KEGG pathway enrichment analysis showed that the apoptotic pathway and multiple carbohydrate metabolism and amino acid metabolism pathways in BmE were enriched,indicating that carbohydrate metabolism,amino acid metabolism and apoptosis signaling pathways played an important role in the process of beauvericin acting on BmE cells.HEK293T is enriched with pathways related to viral infection,metabolism and apoptosis,indicating that beauvericin may induce apoptosis by affecting the energy metabolism of cells when interacting with human cells.It is worth mentioning that the Hippo signaling pathway,apoptosis signaling pathway and MAPK signaling pathway identified in the two cells are all related to apoptosis,so we further verified these pathways,and found that the gene knockout related to the Hippo signaling pathway and the apoptotic signaling pathway can significantly improve the tolerance of cells to beauvericin,and the MAPK signaling pathway plays an important role in the exposure treatment of beauvericin,and it is found that flow cytometry detection beauvericin exposure induces a significant increase in apoptosis in both cells,indicating that beauvericin plays an important role in the toxicological mechanism of different cells.