Mechanism Of Anti-TB Drugs, Isoniazid (INH)-induced Oxidative Damage Of Silkworm.Bombyx Mori

Silkworm （Bombyx mori） is not only a kind of economically important insect, butalso a mode of Lepidoptera insects. It has a lot in commom with human beings on bothgenes and basic metabolism. Compared with fruit flies, silkworms show a higherdegree of differentiation and are produced in a large size. These has provided lifescience a research model which is of modestly complexity and highly practicality. Usingsilkworm as a model animal for drug screening and evaluation has a number ofadvantages: Silkworms and mammals have the same drug-metabolizing enzymes;Silkworms are similar to mammals in drug absorption efficiency of the intestinal canal,sensitivity to pathogens as well as drug effect; Silkworms are in a moderate size,which provides convenience for injection of haemolymph and intestinal canal; Besides,they are produced in low cost and within short generation cycle, which is in accordancewith the3R replacement, kept from the issue of animal ethics; what’s more, silkwormsmove slowly, bringing little biological danger.As model animals, silkworms play a role in human physiology, disease, geneticresearch and development of new drugs. The researches on silkworms has brought theestablishment of innate immune activation mode, bacterial infection mode and liverdisease patterns, which is of great significance in the field of medical research.Isoniazid （INH） is the irreplaceable clinical first-line anti-TB drugs, whose currentmedical damage effect has always been a puzzle in medical circle. For a long time,mammals have been used as liver injury model for research with INH, but there are stillmany problems to be resolved.In order to explore the feasibility of the establishment of silkworm as a drugscreening and toxicity evaluation model, silkworms are selected in this research asexperimental animals of INH function after acute toxicity evaluation, glutathione metabolism and GST Activity transferase activity and gene expression differences in thefat body. The major findings are as follows:1.The day3larvae of the5th intar Dazao silkworm-Bombyx mori wereexperimented by oral injection with some dose gradient isoniazid. The treatment causedan obvious reaction on silkworm larvae to the acute toxicity of INH, showing a varietyof symptoms of getting poisoned; Besides, the INH toxicity tolerance in femalesilkworm larvae is poorer than the male silkworm, which shows a higher toxicitysensitivity in female silkworm larvae; There are differences in the median lethal dose(LD₅₀) of isoniazid between male and female silkworm which also show a bigdifference in the exogenous drug-resistance.2. The anti-tuberculosis drug isoniazid in a dose of2,000mg/kg of was used totreat the day3larvae of the5th intar silkworm, which aimed to detect the antioxidantdetoxification-related metabolic changes in midgut, hemolymph and fat body ofsilkworm. The results indicated that after the silkworm were fed with isoniazid, theglutathione metabolism in male and female silkworm organizations and the ratio ofreduced and oxidized glutathione showed a regular change. Moreover, the changes inthe activity of glutathione S-transferase （GST） reflect the difference in thedetoxification among silkworm. In addition, the GST activity in male silkworm washigher than that in the female silkworm, and fat body is similarly higher than in theintestine. After the silkworm was fed with isoniazid, changes in glutathione redox stateand enzyme activity took place, during which the fat body played a major role ofmetabolic detoxification3. The study compareed the activity’s change of GPT and GOP in the haemolymphorganization of silkworm by the oral injection with isoniazid. The results indicated thatwhen different doses of INH were used to treat the fat body in silkworm, this tissue canproduce different degrees of toxic effects. The damage to the fat body, such as tissuemay result in GPT and GOT being released into haemolymph, so that the GPT and GOTin high activity can be detected. The variation of enzyme activity in male silkworm islower than that in the female silkworm. By the test of GPT and GOT vitality in haemolymph tissue, the degree of damage to the fat body and other tissues with INHpoisoningcan be estimated.4.The day3larvae of the5th intar female silkworm were constructed to a librarybefore and after feeding INH with the digital gene expression spectrum （RNA-Seq）technology. Then we used the two library from control group and experimental group todo expression and distribution analysis with Clean reads, gene expression andannotation, gene expression levels to determine, differences in gene expression andgene ontology function significant enrichment analysis and so on. The results indicatedthat after and before treated with INH, there are75marked different expressiongenes,with44up-regulated and31down-regulated different genes in the tissue of fatbody. The GO function cluster analysis of these genes attributed to metabolic and signaltransduction related biological functions and participate in similar physiologicalprocesses. KEGG Pathy analysis showed that relates to the metabolism, biologicalsynthesis and signal transduction, glutathione metabolism and GPT and GOT gene.These works will help to detoxify silkworm related genes and explore the mechanism ofgene regulatory networks.By INH drug action silkworm larvae preliminary investigation of the physiologicaland biochemical and digital gene expression profiles, this study provides reference forfurther study on silkworm INH molecules detoxification mechanism.