Proteomic profiling of the phosphoproteins in the rat thalamus, hippocampus and frontal lobe after propofol anesthesia

Propofol is widely used in medical procedures such as gastrointestinal endoscopy in outpatient clinics [1], pediatric MRI examinations [2] and pediatric radiotherapy [3] because of its rapid onset, controllable delivery and rapid recovery. Because of its various advantages and wide range of applications, the mechanism of propofol as a general anesthetic has been the focus of increasing scientific research and much attention from anesthesiologists. Nevertheless, the specific mechanism remains unclear.

Thus far, anesthetic drugs have been known to exert their effect mainly by regulating both the synaptic transmission of key parts of the central nervous system and ion channels in the membrane [4]. Both the neurotransmitters that play an important role in synaptic transmission and ion channels are mostly proteins. Protein modifications, especially phosphorylation and dephosphorylation, play key roles in various cellular functions, such as cell differentiation [5], cell growth and apoptosis [6]. Kondratyuk et al. [7] confirmed that depolarization can increase the phosphorylation of sodium channels in a study conducted in rat brain synaptosomes. In addition, abnormal phosphorylation can cause abnormal cellular activities. Studies suggested that abnormal phosphorylation of tau in brain tissue precedes the formation of neurofibrillary tangles in Alzheimer’s disease [8]. Furthermore, increased tau phosphorylation has been reported in animals subjected to isoflurane and desflurane inhalation, which may contribute to the short-term cognitive dysfunction following anesthetic administration [9,10]. Therefore, detecting the changes in phosphorylation after propofol infusion will be of great help in exploring the underlying mechanism of the general anesthetic action of propofol.

The brain is a highly interactive entity, in which a number of separate brain areas cooperate to execute biological functions [11]. The thalamus may be thought as a type of relay and is believed to act as the switchboard of information between a variety of subcortical areas and the cerebral cortex [12]. Additionally, functional brain imaging also confirmed that the thalamus is the key target for anesthetic action [13]. Studies have shown that the hippocampus is responsible for mental behaviors such as initial learning and memory as well as for conscious behavior [14]. Wei H et al. [15] reported that propofol affects LTD expression in hippocampal CA1 dendrites in rats, which was assumed to be the reason for propofol-induced learning and memory damage. The cerebral cortex is the final target of arousal systems, and the dorsolateral prefrontal cortex is one of most important parts of the cerebral cortex, participating in activities such as emotion recognition, voluntary movements and working memory [16] as well as the maintenance of the arousal state in mammals [17]. Thus, phosphorylated proteins in the thalamus, hippocampus and frontal lobes were extracted in an animal model induced by propofol anesthesia, and the proteins that were differentially expressed before and after anesthesia were identified using two-dimensional electrophoresis and mass spectrometry in an attempt to uncover meaningful clues about the anesthetic mechanism of propofol.

Although the anesthetic effect of propofol has been extensively studied by anesthesiologists, the molecular mechanism via which propofol exerts its anesthetic effect remains unknown. Research has demonstrated that decreasing the infusion speed of propofol could effectively decrease the side effects resulting from propofol anesthesia [19]. Larsson et al. [20] also found that the minimum amount of hemodynamic fluctuations and the most stable anesthetic effect occurred when propofol was delivered at a speed of 10 mg/kg/min. Because the anesthetized rats in this experiment were not subjected to any surgical procedures, the infusion speed could be set a lower level (24 mg/kg/h), with which a stable animal model was established.

Futter et al. [21] indicated that 7, 14 and 10 proteins spots were found to be differentially expressed in rat brain tissues 3 h, 24 h and 72 h after propofol anesthesia, respectively. Uniform differentially expressed protein profiles were noticed in the brain tissues of rats exposed to propofol or sevoflurane, and some proteins even showed a trend with the opposite change, indicating that the mechanism of action of inhaled anesthetics was not completely identical to that of intravenous anesthesia [22]. The insoluble protein profiles in the rat hippocampus were successfully identified, and their correlation with cognitive dysfunction after anesthesia was further explored by Xuena Zhang et al. [23]. These experimental results provided a new clue for explaining the molecular mechanisms of postoperative cognitive dysfunction after anesthesia. However, these studies examined total proteins, and protein synthesis is a time-consuming process. In contrast, protein phosphorylation is a fast and reversible process that is involved in a wide variety of crucial cellular activities [6], in which the transcription and synthesis of new proteins are not involved. In all post-translational modification, phosphorylation and dephosphorylation is the most important which regulate all activites of life including cellular signal transduction, cell differentiation, cell growth and apoptosis [5-7]. In addition, the rapid onset of phosphorylation modifications coincides with the onset of the propofol anesthesia. Therefore, the profile of phosphorylated proteins in the thalamus, hippocampus and frontal lobes was determined using proteomics in this study. Using mass spectrometry, sixteen proteins were found to be differentially expressed after propofol anesthesia, and the phosphorylation of keratin 18, gelsolin and apolipoprotein E was measured using Western blot to verify the 2D electrophoresis results.

The identified differentially expressed phosphorylated proteins were subjected to bioinformatics analysis. Six proteins, including keratin 18, gelsolin, tubulin 2c chain, macrophage-capping protein, actin and apolipoprotein E were identified. These proteins form cytoskeletal structures or participate in stabilizing the cytoskeletal structure. Additionally, the changing trends in the phosphorylation levels of the three proteins were verified using Western blots, suggesting that propofol anesthesia can cause changes in cytoskeletal proteins in brain tissue.

The cytoskeleton mainly consists of microtubules, microfilaments and intermediate filaments. The tubulin 2c chain is the main structural component of microtubules, and actin is the main constituent of microfilaments. In addition to maintaining the stability of cell shape and structure, cytoskeletal proteins also regulate various molecular activities such as intracellular transport [24], energy and information transfer [25], and signal transduction. Alterations in cytoskeletal architecture can result in changes in ion channels and account for the occurrence and development of brain-related disorders, which was first found in neurocytes. In 1981, Fukuda et al. [26] reported for the first time that cytoskeletal architecture disruption could inhibit the excitatory functions of sevrral central nervous systems (CNS) component. In 1988, Srinivasan et al., using the voltage-clamp technique [27], confirmed that the cytoskeleton was capable of modulating sodium channels in neurocytes. Propofol-induced amnesia may be related to the down-regulation of activity-regulated cytoskeleton-associated protein (Arc) in the hippocampus. In contrast, the Arc mRNA level does not change significantly, indicating that propofol may exert its function by affecting protein modification [28].

Microtubule-associated proteins (MAPs) bind to the tubulin subunits that form microtubules. Tau proteins are type II MAPs that are abundant in neurons in the central nervous system, and their phosphorylation state can be modulated by a specific set of phosphatases and phosphokinases, which play a vital role in maintaining neuronal function and development. Hyperphosphorylation of the tau protein is assumed to be involved in the neuropathogenesis of several types of dementia, such as Alzheimer’s disease and postoperative cognitive dysfunction [8]. Sevoflurane, isoflurane and propofol can induce tau hyperphosphorylation, which may account for the occurrence of postoperative cognitive dysfunction [10,29]. Reduced apolipoprotein E phosphorylation caused by anesthetic propofol was demonstrated and was later confirmed using Western blot in this experiment. Apolipoprotein E is synthesized and secreted predominantly by astrocytes and microglia in the brain, which participates in neuron repair after injury, dendritic growth and the maintenance of synaptic plasticity [30]. Apolipoprotein E also plays a vital role in the pathogenesis of Alzheimer’s disease, where apolipoprotein E is found in the amyloid plaques and neurofibrillary tangles characteristic of Alzheimer’s disease [31].

The deposition of monomers and polymers of hyperphosphorylated tau protein in the brain of a transgenic mice expressing apolipoprotein E4 (C112R) [32,33] suggested that apolipoprotein E affected tau phosphorylation. We speculate that the postoperative cognitive dysfunction induced by anesthetic propofol may result from alterations in serum apolipoprotein E levels. Nevertheless, the interwoven relationship between these factors and whether apolipoprotein E phosphorylation is involved in this process must be further explored.

Bioinformatics analysis indicated that gelsolin and keratin 18 exhibit responses to ethanol. Ethanol can weaken our body’s reactions to outside stimulation, which is similar to the behavioral changes that emerge after propofol anesthesia. Amino acid neurotransmitter receptors play an important role in alcohol dependence [34]. Ethanol is both a gamma-aminobutyric acid (GABA) receptor agonist and an N-methyl-D-aspartate (NMDA) receptor antagonist, which results in degenerative alterations of the nervous system during brain development by inhibiting ERK phosphorylation [35,36]. The effect of ethanol is similar to that of propofol on brain stem cell apoptosis during brain development. Glutamic acid (Glu) serves as the main neurotransmitter for the inputs and outputs as well as the intrinsic circuitry of the hippocampus, which appears to be the brain region that is most sensitive to alcohol damage [37] and is the target of the anesthetic effect of propofol. In summary, the targets of and transmitters in general anesthetic agents are similar to those of the effect of alcohol on the brain. Furthermore, studies have affirmed that ethanol was able to induce the dephosphorylation of keratin 18 in the liver and kidney [38]. In our study, keratin 18 was also dephosphorylated in the rat hippocampus following propofol administration compared with the controls. We speculated that the proteins responsive to ethanol may also contribute to the anesthetic effect of propofol. However, the specific mechanisms require further study.

In conclusion, 16 differentially expressed phosphorylated proteins in the thalamus, hippocampus and frontal lobes were found using proteomics methods in this study. Additionally, bioinformatics were also used to analyze the common characteristics of the differentially expressed proteins to study the underlying mechanisms of the general anesthetic action. These experimental data will definitely provide meaningful references for the clarification of the mechanism of action of the general anesthetic propofol.