This experiment was conducted to examine the effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone (20-HE) concentration for juvenile Fenneropenaeus chinensis with an...This experiment was conducted to examine the effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone (20-HE) concentration for juvenile Fenneropenaeus chinensis with an initial wet body weight of 1.115 g±0.012 g. The salinity of the control treatment (represented by DO) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The range of salinity fluctuation was 4. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8d, respectively; each original salinity was decreased abruptly to salinity 24, which first lasted for another 2 d and was then raised to its initial value 28. This constituted a salinity fluctuation cycle and afterwards the cycle repeated. It was found that: 1) The weight gain of shrimp in treatment D4 was 202.9% and significantly higher than that in treatment DO (P〈0.05). The molting rate in treatment D2 was the lowest, while that in treatment D6 was the highest and significantly higher than those in treatments DO and D2 (P〈0.05). 2) The hemolymph 20-HE concentration of shrimps in all treatments was at low levels and increased first gradually during the inter-molt period and then increased sharply. It reached a peak value during the pre-molt stage, and then decreased abruptly during the post-molt stage when the lowest level occurred. 3) During the post-molt stage, the hemolymph 20-HE concentration in treatment D2 was 7.47pg μL^-1 and significantly higher than that in treatment DO (P〈0.05). There were, however, no significant differences in hemolymph 20-HE concentration among all five treatments during the inter-molt stage (P〉0.05). During the pre-molt stage, the hemolymph 20-HE concentration in treatment D6 was significantly higher than that in treatment DO (P〈0.05), whereas the hemolymph 20-HE concentration in treatment D2 was significantly lower than that in other treatments (P〈0.05). Adequate salinity fluctuation promoted the molting rate through increasing hemolymph 20-HE concentration at the pre-molt stage.展开更多
The epidermal collagenase activity and its induction by 20-hydroxyecdysone in Uca pugilator were investigated. Zymographic electrophoresis showed four bands of collagenase activity, 16, 19, 22 and 29 kDa in molecular ...The epidermal collagenase activity and its induction by 20-hydroxyecdysone in Uca pugilator were investigated. Zymographic electrophoresis showed four bands of collagenase activity, 16, 19, 22 and 29 kDa in molecular weight, with the former two accounting for 60% and 36% , respectively, of the total collagenase activity. The collagenase activity varies during the molting cycle. Among the molt stages tested, Premolt Stage Do exhibited the highest epidermal collagenase activity for both the 16 and 19 kDa isoenzymes and, as the molt stage proceeded, the enzymatic activity of these two isoenzymes decreased, with the lowest activity for both found in Premolt Stage D3 -4 - Injection of 20-hydroxyecdysone significantly induced the activity of the 16 kDa eollagenase in the epidermis of Uca pugilator, suggesting that the activity of this isoenzyme is under molting hormone control. Although 20-hydroxyeedysone injection did not result in a statistically significant increase in the activity of the 19 kDa isoenzyme, a tendency of the induction was nonetheless demonstrated. This is the first report on epidermal collagenase activity and its induction by the molting hormone in a crustacean .展开更多
Objective:To investigate the anti-proliferative effects of 20-hydroxyecdysone isolated from the bark of Dacrycarpus imbricatus(Blume) de Laub.Methods:Column chromatography was used for isolation of compounds from plan...Objective:To investigate the anti-proliferative effects of 20-hydroxyecdysone isolated from the bark of Dacrycarpus imbricatus(Blume) de Laub.Methods:Column chromatography was used for isolation of compounds from plant material.The structure of the isolated compound was identified by mass spectrometry and nuclear magnetic resonance techniques,including HSQC,HMBC,NOE-difference experiments.The isolated compound was tested for its anti-proliferative activity in acute myeloid leukemia(AML) and OCI-AML cells.Results:Compound 1 was isolated from the ethyl acetate fraction of Dacrycarpus imbricatus barks by column chromatography.Its chemical structure was identified as 20-hydroxyecdysone(20HE),a cholestane-type ecdysteroid,by a combination of mass spectrometry and nuclear magnetic resonance spectrometric analyses.Our goal was to test the anti-proliferative activity of 20 HE using the OCI-AML cell line.20 HE significantly decreased OCI cell number at a concentration of 1 mg/mL,whereas lower concentrations were ineffective.Moreover,this decrease was due to partial blockage of the G_1/S phase of the cell cycle,with a reduction of cells in the G_2M phase,not due to increased apoptosis.Conclusions:This indicates that 20 HE significantly decreases the number of cells in the G_1/S phase of the cell cycle in human AML cells.This is the first time that the anti-proliferative activity of 20 HE against a human tumor cell line has been reported.展开更多
Objective The hippocampus is thought to be a vulnerable target of microwave exposure.The aim of the present study was to investigate whether 20-hydroxyecdysone(20E)acted as a fate regulator of adult rat hippocampal ne...Objective The hippocampus is thought to be a vulnerable target of microwave exposure.The aim of the present study was to investigate whether 20-hydroxyecdysone(20E)acted as a fate regulator of adult rat hippocampal neural stem cells(NSCs).Furthermore,we investigated if 20E attenuated high power microwave(HMP)radiation-induced learning and memory deficits.Methods Sixty male Sprague-Dawley rats were randomly divided into three groups:normal controls,radiation treated,and radiation+20E treated.Rats in the radiation and radiation+20E treatment groups were exposed to HPM radiation from a microwave emission system.The learning and memory abilities of the rats were assessed using the Morris water maze test.Primary adult rat hippocampal NSCs were isolated in vitro and cultured to evaluate their proliferation and differentiation.In addition,hematoxylin&eosin staining,western blotting,and immunofluorescence were used to detect changes in the rat brain and the proliferation and differentiation of the adult rat hippocampal NSCs after HPM radiation exposure.Results The results showed that 20E induced neuronal differentiation of adult hippocampal NSCs from HPM radiation-exposed rats via the Wnt3a/β-catenin signaling pathway in vitro.Furthermore,20E facilitated neurogenesis in the subgranular zone of the rat brain following HPM radiation exposure.Administration of 20E attenuated learning and memory deficits in HPM radiation-exposed rats and frizzled-related protein(FRZB)reduced the 20E-induced nuclear translocation ofβ-catenin,while FRZB treatment also reversed 20E-induced neuronal differentiation of NSCs in vitro.Conclusion These results suggested that 20E was a fate regulator of adult rat hippocampal NSCs,where it played a role in attenuating HPM radiation-induced learning and memory deficits.展开更多
Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understandin...Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly understood.Genome-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host interaction.Previously,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor responses.In this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action.Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell apoptosis.The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to 20E.Our findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.展开更多
The steroid hormone 20-hydroxyecdysone(20E)has been described to regulate fat body lipid metabolism in insects,but its accurate regulatory mechanism,especially the crosstalk between 20E-induced lipid metabolism and gl...The steroid hormone 20-hydroxyecdysone(20E)has been described to regulate fat body lipid metabolism in insects,but its accurate regulatory mechanism,especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear.Here,we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae,a notorious pest in forestry.Lipidomics analysis showed that a total of 1907 lipid species were identified in the fat body of H.cunea larvae assigned to 6 groups and 48 lipid classes.The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples,indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H.cunea larvae.Further studies demonstrated that 20E accelerated fatty acidβ-oxidation,inhibited lipid synthesis,and promoted lipolysis.Meanwhile,the activities of pyruvate carboxylase,phosphoenolpyruvate carboxykinase,fructose-1,6-bisphosphatase,and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H.cunea larvae.As well,the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H.cunea larvae after treatment with 20E.Taken together,our results revealed that 20E disturbed fat body lipid homeostasis,accelerated fatty acidβ-oxidation and promoted lipolysis,but negatively regulated gluconeogenesis in H.cunea larvae.The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.展开更多
The aim of this study was to investigate the possible protective effects and mechanisms of 20-hydroxyecdysone, an insect steroid hormone, on HEO2-induced cytotoxicity in human neuroblastoma SH-SYSY cells. Pretreatment...The aim of this study was to investigate the possible protective effects and mechanisms of 20-hydroxyecdysone, an insect steroid hormone, on HEO2-induced cytotoxicity in human neuroblastoma SH-SYSY cells. Pretreatment with 20-hydroxyecdysone significantly elevated the cell viability and decreased LDH leakage in H2O2-treated SH-SY5Y cells. 20-Hydroxyecdysone also dramatically reduced malondialdehyde (MDA) contents and enhanced the superoxide dismutase (SOD) activities under oxidative stress conditions. Furthermore, 20-hydroxyecdysone pretreatment inhibited apoptosis by decreasing the Bax/Bcl-2 ratio and attenuating the activation of caspase-3. These results suggest that 20-hydroxyecdysone can protect SH-SYSY cells against H2O2-induced cytotoxicity and might potentially be used to treat neurodegenerative diseases induced by oxidative stress and anontosis.展开更多
20E-hydroxyecdysone(20E)plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response.Insect metamorphosis is a highly successful strategy for environme...20E-hydroxyecdysone(20E)plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response.Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens.20E regulates a series of antimicrobial peptides(AMPs)through the immunodeficiency(IMD)pathway activation in Drosophila;nevertheless,whether other immune pathways are involved in 20E-regulated insect immunity is unknown.Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition(ML)family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway.In this study,we further demonstrate that BmMD-2A is also positively regulated by 20E,and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors,the AMP genes against Escherichia coli and Staphylococcus aureus,through the regulation of BmMD-2A in larval metamorphosis,implying that B.mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage,which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.展开更多
In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N-acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene Lm- CHS1, are expressed mainly in the integument and are responsible f...In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N-acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene Lm- CHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20-hydroxyecdysone (20E) is still largely unclear. Here, we showed the developmental expression pattern ofLmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference (RNAi) directed toward a common region of the two isoforms of LmEcR (LmEcRcom) reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection-based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late-response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi-based pest control.展开更多
Juvenile hormone(JH) and 20-hydroxyecdysone(20 E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant(Met) and germ-cell expressed(Gce), have been identi...Juvenile hormone(JH) and 20-hydroxyecdysone(20 E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant(Met) and germ-cell expressed(Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases.Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C(PKC) which phosphorylated ultraspiracle(USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20 E through its nuclear receptor complex Ec RUSP. The usp;mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20 E signaling that delayed developmental timing. The usp;mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20 E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.展开更多
Mermithid nematodes,such as Ovomermis sinensis,are used as biological control agents against many insect pests,including cotton bollworm(Helicoverpa armigera).However,given the host's robust immune system,the infe...Mermithid nematodes,such as Ovomermis sinensis,are used as biological control agents against many insect pests,including cotton bollworm(Helicoverpa armigera).However,given the host's robust immune system,the infection rate of O.sinensis is low,thus restricting its widespread use.To understand the host defense mechanisms against mermithid nematodes,we identified and characterized a protein involved in the recognition of O.sinensis,the potential O.sinensis-binding protein C-type lectin 1(HaCTLla and/or HaCTLIb),which was eluted from the surface of O.sinensis after incubation with H.armigera plasma.HaCTLIb is homologous to the previously reported HaCTLla protein.HaCTLI was predominantly expressed in hemocytes and was induced by the steroid hormone 20-hydroxyecdysone through ecdysone receptor(HaEcR)or ultraspiracle(HaUSP),or both.Binding assays confirmed the interactions of the HaCTLI proteins with O.sinensis but not with Romanomermis wuchangensis,a parasitic nematode of mosquito.Moreover,the HaCTL1 proteins were secreted into the hemocoel and promoted hemocyte-mediated encapsulation and phagocytosis.A knockdown of HaEcR and/or HaUSP resulted in compromised encapsulation and phagocytosis.Thus,HaCTLI appears to modulate cellular immunity in the defense against parasitic nematodes,and the 20-hydroxyecdysone-HaEcR-HaUSP complex is involved in regulating the process.展开更多
20-Hydroxyecdysone(20E)derived from plants has a wide range of physiological and pharmacological ef ects on animals and humans,and rapid and sensitive methods for screening of the endogenous 20E in plants are thus req...20-Hydroxyecdysone(20E)derived from plants has a wide range of physiological and pharmacological ef ects on animals and humans,and rapid and sensitive methods for screening of the endogenous 20E in plants are thus required.Herein,a matrixassisted laser desorption/ionization time-of-f ight tandem mass spectrometry(MALDI-TOF/TOF MS)method is described for rapid and sensitive determination of endogenous 20E in plants.It is based on the use of the(3-(acridin-9-ylamino)phenyl)boronic acid(AYPBA)as the mass tag to assist the MS and tandem MS(MS^(n))analysis of 20E on MALDI-TOF/TOF MS.Good linearity was obtained with a determination coef cient(R^(2))larger than 0.99 in the range of 0.025–2.5μΜ.The limit of detection(LOD)was 2.4 fmol.Acceptable precision and accuracy were gained by intra-and inter-day analysis with relative standard deviations less than 19.5%and relative recoveries ranging from 85.7 to 105.2%.In addition,the AYPBA labeled 20E produced abundant characteristic fragment ions under the high energy collision-induced dissociation,which facilitated the identif cation of 20E by MS^(2)analysis on MALDI-TOF/TOF MS.Using the method,we enabled the identif cation and quantif cation of endogenous 20E in four herbs including Cyanotis arachoidea,Achyranthes bidentata,Spinacia oleracea and Chenopodium quinoa willd.,demonstrating the feasibility of the proposed method for screening of the endogenous 20E in plants.展开更多
基金supported by funds from the National Natural Science Foundation of China (Grant No. 30571441)the Project of Reward Research Foundation for Talented Young and Middle Aged Scientists of Shandong, China (Grant No. 2006BS07002)the National Great Project of Scientific and Technical Supporting Programs Funded by the Ministry of Science and Technology of China in the 11th Five-Year Plan (Grant No. 2006BAD09A07)
文摘This experiment was conducted to examine the effects of salinity fluctuation frequency on the growth, molting rate and hemolymph 20-hydroxyecdysone (20-HE) concentration for juvenile Fenneropenaeus chinensis with an initial wet body weight of 1.115 g±0.012 g. The salinity of the control treatment (represented by DO) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The range of salinity fluctuation was 4. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8d, respectively; each original salinity was decreased abruptly to salinity 24, which first lasted for another 2 d and was then raised to its initial value 28. This constituted a salinity fluctuation cycle and afterwards the cycle repeated. It was found that: 1) The weight gain of shrimp in treatment D4 was 202.9% and significantly higher than that in treatment DO (P〈0.05). The molting rate in treatment D2 was the lowest, while that in treatment D6 was the highest and significantly higher than those in treatments DO and D2 (P〈0.05). 2) The hemolymph 20-HE concentration of shrimps in all treatments was at low levels and increased first gradually during the inter-molt period and then increased sharply. It reached a peak value during the pre-molt stage, and then decreased abruptly during the post-molt stage when the lowest level occurred. 3) During the post-molt stage, the hemolymph 20-HE concentration in treatment D2 was 7.47pg μL^-1 and significantly higher than that in treatment DO (P〈0.05). There were, however, no significant differences in hemolymph 20-HE concentration among all five treatments during the inter-molt stage (P〉0.05). During the pre-molt stage, the hemolymph 20-HE concentration in treatment D6 was significantly higher than that in treatment DO (P〈0.05), whereas the hemolymph 20-HE concentration in treatment D2 was significantly lower than that in other treatments (P〈0.05). Adequate salinity fluctuation promoted the molting rate through increasing hemolymph 20-HE concentration at the pre-molt stage.
文摘The epidermal collagenase activity and its induction by 20-hydroxyecdysone in Uca pugilator were investigated. Zymographic electrophoresis showed four bands of collagenase activity, 16, 19, 22 and 29 kDa in molecular weight, with the former two accounting for 60% and 36% , respectively, of the total collagenase activity. The collagenase activity varies during the molting cycle. Among the molt stages tested, Premolt Stage Do exhibited the highest epidermal collagenase activity for both the 16 and 19 kDa isoenzymes and, as the molt stage proceeded, the enzymatic activity of these two isoenzymes decreased, with the lowest activity for both found in Premolt Stage D3 -4 - Injection of 20-hydroxyecdysone significantly induced the activity of the 16 kDa eollagenase in the epidermis of Uca pugilator, suggesting that the activity of this isoenzyme is under molting hormone control. Although 20-hydroxyeedysone injection did not result in a statistically significant increase in the activity of the 19 kDa isoenzyme, a tendency of the induction was nonetheless demonstrated. This is the first report on epidermal collagenase activity and its induction by the molting hormone in a crustacean .
基金the National Foundation for Science & Technology Development(NAFOSTED) of Vietnam for financial support of this research(code:104.01-2013.62)the Italian Ministery of Foreign Affairs and International Cooperation(MAECI)
文摘Objective:To investigate the anti-proliferative effects of 20-hydroxyecdysone isolated from the bark of Dacrycarpus imbricatus(Blume) de Laub.Methods:Column chromatography was used for isolation of compounds from plant material.The structure of the isolated compound was identified by mass spectrometry and nuclear magnetic resonance techniques,including HSQC,HMBC,NOE-difference experiments.The isolated compound was tested for its anti-proliferative activity in acute myeloid leukemia(AML) and OCI-AML cells.Results:Compound 1 was isolated from the ethyl acetate fraction of Dacrycarpus imbricatus barks by column chromatography.Its chemical structure was identified as 20-hydroxyecdysone(20HE),a cholestane-type ecdysteroid,by a combination of mass spectrometry and nuclear magnetic resonance spectrometric analyses.Our goal was to test the anti-proliferative activity of 20 HE using the OCI-AML cell line.20 HE significantly decreased OCI cell number at a concentration of 1 mg/mL,whereas lower concentrations were ineffective.Moreover,this decrease was due to partial blockage of the G_1/S phase of the cell cycle,with a reduction of cells in the G_2M phase,not due to increased apoptosis.Conclusions:This indicates that 20 HE significantly decreases the number of cells in the G_1/S phase of the cell cycle in human AML cells.This is the first time that the anti-proliferative activity of 20 HE against a human tumor cell line has been reported.
基金supported by grants from the National Natural Science Fund[NO.2015AA8092018A]the Natural Science Foundation of Chongqing municipality[NO.cstc2018jcyjAX0186]
文摘Objective The hippocampus is thought to be a vulnerable target of microwave exposure.The aim of the present study was to investigate whether 20-hydroxyecdysone(20E)acted as a fate regulator of adult rat hippocampal neural stem cells(NSCs).Furthermore,we investigated if 20E attenuated high power microwave(HMP)radiation-induced learning and memory deficits.Methods Sixty male Sprague-Dawley rats were randomly divided into three groups:normal controls,radiation treated,and radiation+20E treated.Rats in the radiation and radiation+20E treatment groups were exposed to HPM radiation from a microwave emission system.The learning and memory abilities of the rats were assessed using the Morris water maze test.Primary adult rat hippocampal NSCs were isolated in vitro and cultured to evaluate their proliferation and differentiation.In addition,hematoxylin&eosin staining,western blotting,and immunofluorescence were used to detect changes in the rat brain and the proliferation and differentiation of the adult rat hippocampal NSCs after HPM radiation exposure.Results The results showed that 20E induced neuronal differentiation of adult hippocampal NSCs from HPM radiation-exposed rats via the Wnt3a/β-catenin signaling pathway in vitro.Furthermore,20E facilitated neurogenesis in the subgranular zone of the rat brain following HPM radiation exposure.Administration of 20E attenuated learning and memory deficits in HPM radiation-exposed rats and frizzled-related protein(FRZB)reduced the 20E-induced nuclear translocation ofβ-catenin,while FRZB treatment also reversed 20E-induced neuronal differentiation of NSCs in vitro.Conclusion These results suggested that 20E was a fate regulator of adult rat hippocampal NSCs,where it played a role in attenuating HPM radiation-induced learning and memory deficits.
基金This work was supported by grants from the National Natural Science Foundation of China(No.32122084)Chongqing Natural Science Foundation(No.cstc2021ycjh-bgzxm0005)+1 种基金PhD Start-Up Foundation of Southwest University(No.SWU120012)Fundamental Research Funds for the Central Universities(No.SWU-KT22042).None of these fundings played any role in the design of the study,collection,analysis,or interpretation of data or in the writing of the manuscript.
文摘Metamorphosis is a complex developmental process involving multiple pathways and a large number of genes that are regulated by juvenile hormone(JH)and 20-hydroxyecdysone(20E).Despite important progress in understanding various aspects of silkworm biology,the hormone signaling pathway in the silkworm remains poorly understood.Genome-wide screening using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-based libraries has recently emerged as a novel method for analyzing genome function,enabling further research into essential genes,drug targets,and virus-host interaction.Previously,we constructed a genome-wide CRISPR/Cas9-based library of the silkworm(Bombyx mori)and successfully revealed the genes involved in biotic or abiotic stress factor responses.In this study,we used our silkworm CRISPR library and large-scale genome-wide screening to analyze the key genes in the silkworm 20E signaling pathway and their mechanisms of action.Functional annotation showed that 20E regulates key proteins in processes that mainly occur in the cytoplasm and nucleus.Pathway enrichment analysis showed that 20E can activate phosphorylation and may affect innate immunity,interfere with intracellular nutrition and energy metabolism,and eventually cause cell apoptosis.The screening results were experimentally validated by generating cells with knockout alleles of the relevant genes,which had increased tolerance to 20E.Our findings provide a panoramic overview of signaling in response to 20E in the silkworm,underscoring the utility of genome-wide CRISPR mutant libraries in deciphering hormone signaling pathways and the mechanisms that regulate metamorphosis in insects.
基金the Natural Science Foundation of Heilongjiang Province(LH2021C010)National Undergraduate Training Programs for Innovations(202110225005)Fundamental Research Fund for the Central Universities(2572022DS09,2572020DR09,2572020DR02).
文摘The steroid hormone 20-hydroxyecdysone(20E)has been described to regulate fat body lipid metabolism in insects,but its accurate regulatory mechanism,especially the crosstalk between 20E-induced lipid metabolism and gluconeogenesis remains largely unclear.Here,we specially investigated the effect of 20E on lipid metabolism and gluconeogenesis in the fat body of Hyphantria cunea larvae,a notorious pest in forestry.Lipidomics analysis showed that a total of 1907 lipid species were identified in the fat body of H.cunea larvae assigned to 6 groups and 48 lipid classes.The differentially abundant lipids analysis showed a significant difference between 20E-treated and control samples,indicating that 20E caused a remarkable alteration of lipidomics profiles in the fat body of H.cunea larvae.Further studies demonstrated that 20E accelerated fatty acidβ-oxidation,inhibited lipid synthesis,and promoted lipolysis.Meanwhile,the activities of pyruvate carboxylase,phosphoenolpyruvate carboxykinase,fructose-1,6-bisphosphatase,and glucose-6-phosphatase were dramatically suppressed by 20E in the fat body of H.cunea larvae.As well,the transcriptions of genes encoding these 4 rate-limiting gluconeogenic enzymes were significantly downregulated in the fat body of H.cunea larvae after treatment with 20E.Taken together,our results revealed that 20E disturbed fat body lipid homeostasis,accelerated fatty acidβ-oxidation and promoted lipolysis,but negatively regulated gluconeogenesis in H.cunea larvae.The findings might provide a new insight into hormonal regulation of glucose and lipid metabolism in insect fat body.
文摘The aim of this study was to investigate the possible protective effects and mechanisms of 20-hydroxyecdysone, an insect steroid hormone, on HEO2-induced cytotoxicity in human neuroblastoma SH-SYSY cells. Pretreatment with 20-hydroxyecdysone significantly elevated the cell viability and decreased LDH leakage in H2O2-treated SH-SY5Y cells. 20-Hydroxyecdysone also dramatically reduced malondialdehyde (MDA) contents and enhanced the superoxide dismutase (SOD) activities under oxidative stress conditions. Furthermore, 20-hydroxyecdysone pretreatment inhibited apoptosis by decreasing the Bax/Bcl-2 ratio and attenuating the activation of caspase-3. These results suggest that 20-hydroxyecdysone can protect SH-SYSY cells against H2O2-induced cytotoxicity and might potentially be used to treat neurodegenerative diseases induced by oxidative stress and anontosis.
基金funded by grants from the National Science Foundation of China(grant no.31970462)the Natural Science Foundation of Guangdong Province(no.2020A1515110805,no.2020A1515010867,no.2021A1515012090).
文摘20E-hydroxyecdysone(20E)plays important roles in larval molting and metamorphosis in insects and is also involved in the insect innate immune response.Insect metamorphosis is a highly successful strategy for environmental adaptation and is the most vulnerable stage during which the insect is susceptible to various pathogens.20E regulates a series of antimicrobial peptides(AMPs)through the immunodeficiency(IMD)pathway activation in Drosophila;nevertheless,whether other immune pathways are involved in 20E-regulated insect immunity is unknown.Our previous studies showed that BmMD-2A is a member of the MD-2-related lipid recognition(ML)family of proteins that are involved in the Bombyx mori innate immunity Toll signaling pathway.In this study,we further demonstrate that BmMD-2A is also positively regulated by 20E,and the BmMD-2A neutralization experiment suggested that 20E activates some downstream immune effect factors,the AMP genes against Escherichia coli and Staphylococcus aureus,through the regulation of BmMD-2A in larval metamorphosis,implying that B.mori may use the Toll-ML signaling pathway to maintain innate immune balance in the larval-pupal metamorphosis stage,which is a different innate immunity pathway regulated by 20E compared to the IMD pathway in Drosophila.
文摘In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N-acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene Lm- CHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20-hydroxyecdysone (20E) is still largely unclear. Here, we showed the developmental expression pattern ofLmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference (RNAi) directed toward a common region of the two isoforms of LmEcR (LmEcRcom) reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection-based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late-response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi-based pest control.
基金supported by the National Natural Science Foundation of China(31620103917 31970459 32070441 31702054 and 31930014)the Shenzhen Science and Technology Program(20180411143628272)the Natural Science Foundation of Guangdong Province(2019A1515011899)。
文摘Juvenile hormone(JH) and 20-hydroxyecdysone(20 E) coordinately regulate development and metamorphosis in insects. Two JH intracellular receptors, methoprene-tolerant(Met) and germ-cell expressed(Gce), have been identified in the fruit fly Drosophila melanogaster. To investigate JH membrane signaling pathway without the interference from JH intracellular signaling, we characterized phosphoproteome profiles of the Met gce double mutant in the absence or presence of JH in both chronic and acute phases.Functioning through a potential receptor tyrosine kinase and phospholipase C pathway, JH membrane signaling activated protein kinase C(PKC) which phosphorylated ultraspiracle(USP) at Ser35, the PKC phosphorylation site required for the maximal action of 20 E through its nuclear receptor complex Ec RUSP. The usp;mutant, in which Ser was replaced with Ala at position 35 by genome editing, showed decreased expression of Halloween genes that are responsible for ecdysone biosynthesis and thus attenuated 20 E signaling that delayed developmental timing. The usp;mutant also showed lower Yorkie activity that reduced body size. Altogether, JH membrane signaling phosphorylates USP at Ser35 and thus potentiates 20 E action that regulates the normal fly development. This study helps better understand the complex JH signaling network.
基金the National Natural Science Foundation of China(Grant Nos.31872301 and 31772220)the Fundamental Research Funds for the Central Universities(Grant No.CCNU19TS026).
文摘Mermithid nematodes,such as Ovomermis sinensis,are used as biological control agents against many insect pests,including cotton bollworm(Helicoverpa armigera).However,given the host's robust immune system,the infection rate of O.sinensis is low,thus restricting its widespread use.To understand the host defense mechanisms against mermithid nematodes,we identified and characterized a protein involved in the recognition of O.sinensis,the potential O.sinensis-binding protein C-type lectin 1(HaCTLla and/or HaCTLIb),which was eluted from the surface of O.sinensis after incubation with H.armigera plasma.HaCTLIb is homologous to the previously reported HaCTLla protein.HaCTLI was predominantly expressed in hemocytes and was induced by the steroid hormone 20-hydroxyecdysone through ecdysone receptor(HaEcR)or ultraspiracle(HaUSP),or both.Binding assays confirmed the interactions of the HaCTLI proteins with O.sinensis but not with Romanomermis wuchangensis,a parasitic nematode of mosquito.Moreover,the HaCTL1 proteins were secreted into the hemocoel and promoted hemocyte-mediated encapsulation and phagocytosis.A knockdown of HaEcR and/or HaUSP resulted in compromised encapsulation and phagocytosis.Thus,HaCTLI appears to modulate cellular immunity in the defense against parasitic nematodes,and the 20-hydroxyecdysone-HaEcR-HaUSP complex is involved in regulating the process.
基金supported by the National Key R&D Program of China(2017YFC0906800)the National Natural Science Foundation of China(21635006,31670373 and 21721005)
文摘20-Hydroxyecdysone(20E)derived from plants has a wide range of physiological and pharmacological ef ects on animals and humans,and rapid and sensitive methods for screening of the endogenous 20E in plants are thus required.Herein,a matrixassisted laser desorption/ionization time-of-f ight tandem mass spectrometry(MALDI-TOF/TOF MS)method is described for rapid and sensitive determination of endogenous 20E in plants.It is based on the use of the(3-(acridin-9-ylamino)phenyl)boronic acid(AYPBA)as the mass tag to assist the MS and tandem MS(MS^(n))analysis of 20E on MALDI-TOF/TOF MS.Good linearity was obtained with a determination coef cient(R^(2))larger than 0.99 in the range of 0.025–2.5μΜ.The limit of detection(LOD)was 2.4 fmol.Acceptable precision and accuracy were gained by intra-and inter-day analysis with relative standard deviations less than 19.5%and relative recoveries ranging from 85.7 to 105.2%.In addition,the AYPBA labeled 20E produced abundant characteristic fragment ions under the high energy collision-induced dissociation,which facilitated the identif cation of 20E by MS^(2)analysis on MALDI-TOF/TOF MS.Using the method,we enabled the identif cation and quantif cation of endogenous 20E in four herbs including Cyanotis arachoidea,Achyranthes bidentata,Spinacia oleracea and Chenopodium quinoa willd.,demonstrating the feasibility of the proposed method for screening of the endogenous 20E in plants.