| Whitmania pigra is an important medicinal resource that is widely farmed in aquaculture in China.Hibernation of W.pigra usually begins in November and lasts until next April,and renews when the environmental temperature rises to a high degree,but a decrease in body weight occurring during hibernation and hibernation lasting long time has serious impacts on production efficiency in aquaculture system.Hibernation is important for surviving long term low temperatures has been reported for multiple endotherms.Hibernation in endotherms and ectotherms is characterized by an energy-conserving metabolic depression due to low body temperatures.There are two major issues that must be dealt with to support long-term cell survival during hibernation.First,metabolic and physiological adjustments must be made that support the metabolic demands of a tissue or organism using alternate or at least altered metabolic pathways and substrates.Second,survival of hibernation requires specific alterations that avoid immediate and delayed cell death during both entry and exit from a state of metabolic depression during hibernation cycle.Unlike endotherms hibernation,ectotherms hibernation does not accompany interbout arousal,during which endotherms rewarm to normothermia within a few hours.Due to the limited data of the basic research data of leech,this study first studied the energy metabolism and self-protection mechanism of leech during the hibernation cycle,and improved the basic scientific research of leech hibernation,and then through differential proteomics and phosphorylation proteomics to compare the expression of protein and differential protein phosphorylation in the leech hibernation before and after hibernation,and to study the hibernation process of the leech in detail.This paper is mainly composed of four parts,namely:1.A significant feature of hibernating animals is the inhibition of energy metabolism,and this inhibition is quickly lifted when the animal is awake from hibernation.In this study,we found that the energy metabolism in the hibernation cycle of the leech was significantly changed by detecting the enzyme activity and expression of the key enzymes in the energy metabolism of the leech at the five different periods of hibernation.The results showed that the key enzymes(HK,PK,PDH,IDH,SDH,MDH,UQCR and COX)significantly decreased,the contents of ATP,AMP,ADP,NAD+and NADH decreased during the deep hibernation.The enzyme activity and expression of the key enzymes increased and the contents of ATP,AMP,ADP,NAD+and NADH increased during early arousal.These results indicate that the energy metabolism process(glycolysis,tricarboxylic acid cycle and oxidative phosphorylation process)of leech during hibernation significantly decreased,and the yield of ATP reduced.The energy metabolism rapidly restored and the yield of ATP increased during early arousal.Therefore,the leech hibernating period has a strict regulation system to ensure the supply of energy metabolism during each period.2.Through the detection of intestinal antioxidant enzyme activity and expression in five different periods of leech,we found that leech was inhibited during physiological activity during the deep hibernation,and the antioxidant capacity decreased(enzyme activity and expression decreased).As a result of low temperature stress,antioxidant capacity increased;hibernation by reactive oxygen stress,antioxidant capacity also increased.These results indicate that different stages of the hibernating period have different antioxidant systems.By measuring the enzyme activity and expression of Caspase3 and Caspase8,and combining the expression of apoptotic induction factor AIF1 and anti-apoptotic protein bcl-2,the changes of apoptosis of intestinal tissue during hibernating period were revealed.The results showed that the apoptosis of the intestinal tissue during hibernating period was inhibited,and the cell apoptosis increased during entering into the hibernation and early arousal.In this experiment,the expression of the intestinal HSP27,HSP60 and HSP70 in the deep hibernating period of the leech was significantly higher than that in normal period.During entering into the hibernation,the expression of three heat shock proteins increased.During early arousal,the expression levels of the three HSPs were significantly higher than those in normal period,and the HSP27 hibernation period was significantly higher than that of other periods.Because heat shock protein is a protective protein that improves cell stress,heat shock proteins may provide protective cells during leech hibernating.3.In this study,we applied Tandem mass tags-coupled two-dimensional Liquid chromatography tandem-mass spectrometry to identify and quantify the global proteome expression profile over the hibernation-arousal cycle of W.pigra.A total of 3580 proteins were identified,among which 2184 proteins were quantified and compared throughout the hibernation cycles revealing 390 differentially expressed proteins.Bioinformatics analysis showed that the associated protein associated with fat decomposition was elevated and carbohydrate and protein decomposition protein decreased during deep hibernating,indicating the metabolism of fat metabolism increased,the decomposition of sugar and protein was inhibited during hibernating.In addition,protein synthesis,energy metabolism and transmembrane transport-related proteins were significantly reduced during deep hibernation,while cytoskeletal proteins and heat shock protein-related proteins increased.We hypothesized that leech may weaken transmembrane transport and protein synthesis during hibernation to reduce energy demand,increase cytoskeleton reorganization of the cytoskeleton protein structure stability of cell to protect the intestinal cells.4.The present study aimed to investigate the regulatory mechanisms of hibernation W.pigra,an excellent model ectotherm,and improve the research of hibernation regulation mechanism.3474 phosphorylation sites in 2596 proteins were identified,among which 2598 sites in 1388 proteins were quantified.Different phosphorylation protein constituted the leech protein phosphorylation mutual mapping,involving in signal transduction,energy metabolism,protein synthesis,cell protection,etc.These processes involved in self-protection of the organization during leech hibernate.Test confirmed the activity of related protein phosphorylation changes on the activity of these proteins.These changes of reversible protein phosphorylation on protein phosphorylation cascade controled downstream activity,controling different physiological activities to adapt to different environment in the different hibernation stages.In summary,the study of the energy metabolism and self-protection mechanism of leech during the hibernation cycle can be found that the energy metabolism of the leech obviously inhibited and the antioxidant capacity decreased during deep hibernation,but the heat shock protein rised.Due to low temperature stress,antioxidant capacity and heat shock protein rised during entering into the hibernation.Due to oxidative phosphorylation increasing significantly during early arousal resulting in a large number of reactive oxygen species,antioxidant capacity and heat shock protein rised.Through the analysis of the difference of protein expression and protein phosphorylation level in different periods of leech hibernation found that differences in protein and phosphorylated proteins are involved in energy production,protein synthesis,transcription and translation,anti stress,homeostasis,etc.These processes associated with energy metabolism and cellular self-protection,further suggested leech can regulate energy metabolism and cell self protection mechanisms to adapt to bad environment during hibernation. |
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