Functional Study Of BmFoxO In Response To High Temperature Stress In Silkworm (Bombyx Mori)

Any organism is active within a certain temperature range,and temperature is one of the most obvious environmental factors for biological impact.Temperature can affect the living state,habitat distribution,sex determination,growth and survival of living things.Extreme temperature can cause damage to organisms.For example,high temperature can cause the living organism to be in a state of stress,metabolic disorders occur,and reactive oxygen species abnormally and rapidly increase,which damages nucleic acids,lipids and proteins in cells,resulting in imbalance of redox,oxidative stress response,destroying normal physiological functionsin the organism.The antioxidant enzymes in the organism mainly include superoxide dismutase?SOD?,catalase?CAT?and glutathione peroxidase?Gpx?,etc.,which maintain the oxidative balance in the organism,and these enzymes will be correspondingly Regulation of genes.Fox O is a member of the Forkhead family of transcription factors and is found in a wide variety of eukaryotic organisms and is involved in a variety of biological functions including anti-oxidative stress,apoptosis and autophagy,DNA damage and repair,cell cycle and energy metabolism.It has been reported that Fox O₃ a in the mammalian Fox O family regulates manganese superoxide dismutase?Mn SOD?to eliminate intracellular ROS and thereby reduce cellular oxidative damage,indicating that Fox O is involved in the regulation of oxidative stress in mammals.Silkworm is a long-established economic animal and has made outstanding contributions to the economic development of the Chinese nation and the spread of silk culture.The completion of the "Trilogy" of the silkworm genome project marked by "framework,fine map and genetic variation map" promoted the establishment of a key technology platform for the research and application of functional genes such as silkworm transgenic.Bombyx mori belongs to the lepidopteran model insects and is a variable temperature animal.The external temperature changes play a key role in the growth and development of the silkworm.High temperature treatment leads to a decrease in the survival rate of silkworm,which is related to the increase of ROS in the body.China's sericulture is mainly in the high temperature areas in the south.It is of great significance to study the high temperature resistance mechanism of silkworm and the cultivation of high temperature resistant varieties for the development of sericulture.However,whether Bm Fox O is involved in the regulation of oxidative stress caused by high temperature stress and how to regulate it in silkworm has not been reported.Therefore,this study firstly clarifies the molecular changes and damage caused by high temperature or H₂O₂ oxidative stress in silkworm,which is overexpressed in Bm E cells.Bm Fox O was used to investigate its effects on high temperature and oxidative stress.Finally,Bm E cells were used as the object to elucidate the molecular mechanism of Bm Fox O regulation of oxidative stress in silkworm.The main findings are as follows: 1.Bombyx mori responds to high temperature or H₂O₂-induced oxidative stressThe silkworm was treated with high temperature at 37 ?.Compared with the control at 25 ?,the ROS content in the fat body increased gradually.The expression level of oxidative stress-related genes in the fat body of the silkworm was detected by real-time PCR.The results showed that Bm Fox O was treated within a certain period of time under high temperature stress treatment.The antioxidant enzyme-related genes Bm SOD1,Bm SOD2,Bm SOD3,Bm CAT and Bm GADD45 genes were up-regulated,while the antioxidant enzyme Bm Gpx was down-regulated.The results of physiological and biochemical kits showed that the activity of SOD and CAT increased first and then decreased.Bm Fox O and antioxidant enzyme-related genes showed a consistent trend with high temperature treatment within a certain period of H₂O₂ stress treatment.The results of physiological and biochemical kits showed that the ROS content increased gradually,while the SOD and CAT enzyme activities also showed a consistent trend with high temperature treatment.The above results indicate that high temperature or H₂O₂ induces oxidative stress in silkworm,and Bm Fox O is up-regulated.2.Bm Fox O resists oxidative stress damage in Bm E cellsTo explore the mechanism of high temperature or H₂O₂-induced oxidative damage,we first verified high temperature or H₂O₂ in Bm E cells to cause oxidative damage and explore its mechanism on cells.The CCK-8 kit was used to detect the changes of viability of Bm E cells after treatment at 37 ? for 0,12,24 and 48 h.The results showed that the cell viability decreased with the prolongation of treatment time,and the cells were detected by DCFH-DA probe.After 12 h treatment at ?,the ROS content increased significantly.The results of real-time PCR showed that the expression levels of oxidative stress-related transcription factors and antioxidant enzyme genes?except Bm Gpx?were up-regulated after high temperature induction.The results of physiological and biochemical kits showed that the activity of SOD and CAT increased,and the contents of MDA and H₂O₂ increased significantly after high temperature treatment.The results showed that high temperature stress induced oxidative stress in Bm E cells.High temperature and H₂O₂ have the same trend of oxidative damage in silkworm.In order to better explain the molecular mechanism of oxidative damage,we chose H₂O₂ treatment for subsequent experiments.After treatment with H₂O₂ at a final concentration of 0,250,500,1000,2000 ?M,the viability of the cells was detected.It was found that the cell viability was irreversibly decreased after H₂O₂ treatment at a concentration of 1000 ?M or more,and the ROS content was significantly increased.With the increase of H₂O₂ concentration,the expression levels of Bm Fox O,Bm SOD1,Bm SOD2,Bm SOD3,Bm CAT and Bm GADD45 genes increased gradually,which was consistent with the expression trend in silkworm.SOD and CAT activities and MDA levels continue to rise.In order to explore the function of Bm Fox O in oxidative stress,Bm Fox O overexpression vector was constructed and transfected into cells.It was found that the cells overexpressing Bm Fox O significantly decreased ROS content after high temperature or H₂O₂ treatment compared with the control group.DNA damage and mortality were significantly reduced.The above results indicate that oxidative damage caused by high temperature or H₂O₂ treatment of Bm E cells is consistent with that of silkworm,and overexpression of Bm Fox O can reduce oxidative stress damage of Bm E cells.3.The mechanism of Bm Fox O involved in oxidative stress in silkwormFox O is the major substrate for protein kinase B?PKB or Akt?and its activity depends on the level of phosphorylation.Insulin and growth factors will localize Fox O to the cytoplasm and eventually degrade,while phosphorylated Fox O will localize to the nucleus to regulate the expression of downstream target genes.In order to clarify the activity of Bm Fox O after high temperature and H₂O₂ treatment,we examined the subcellular localization of Bm Fox O and constructed a Bm Fox Ooverexpression vector with EGFP.After transfecting the cells,it was found that the localization of Bm Fox O at 37 ? or H₂O₂ treatment.From the cytoplasm to the nucleus,Western blot results showed that Bm Fox O was detected in the nuclear protein after H₂O₂ stress,suggesting that Bm Fox O in the nucleus is a non-phosphorylated form.Furthermore,the phosphorylation level of Akt in the cells was decreased after H₂O₂ treatment,indicating that H₂O₂ inhibited the phosphorylation level of Akt protein.To explore the transcriptional regulation of Bm Fox O,we analyzed the oxidase gene promoter sequence and predicted the binding motif of the Forkhead transcription factor.In order to clarify whether Bm Fox O directly regulates these genes,primers were designed and a dual fluorescein reporter vector was constructed.Double luciferase activity assay showed that Bm Fox O up-regulates the promoter activity of these antioxidant enzyme genes,and the constitutive form of Bm Fox OCA against these promoters,and the effect of the activity is more obvious.In order to further determine the regulation of Bm Fox O against oxidative genes,biotin-labeled probes were designed.EMSA experiments showed that Bm Fox O can specifically bind to CRE3 probes.In summary,H₂O₂ inhibits Akt phosphorylation and localizes Bm Fox O to the nucleus,thereby enhancing the transcriptional activity of the antioxidant enzyme gene,thereby eliminating intracellular ROS and reducing oxidative damage.