Abstract
Mongolian medical warm acupuncture (MMWA) therapy is a traditional external therapy method that has a definite clinical effect in the treatment of insomnia, but research on its underlying neural mechanisms remains limited. iTRAQ-based quantitative proteomics was used to identify proteins that are potential neural molecules involved in the treatment of insomnia by MMWA. In this study, 6383 proteins were identified, including 45 proteins with increased expression and 31 proteins with decreased expression in the insomnia model group (M) compared to the control group (C); 101 proteins showed an increase and 48 proteins showed a decrease between the warm acupuncture group (W) and C; 26 proteins showed an increase and 22 proteins showed a decrease between W and M. GO and KEGG analysis showed that MMWA treatment of insomnia was closely related to metabolism and hormone synthesis. Differentially expressed proteins included albumin (ALB), Pro-MCH (PMCH), acetoacetyl-CoA synthetase (AACS), N-ethylmaleimide sensitive factor (NSF), potassium/sodium hyperpolarization-activated cyclic-nucleotide gated channel 4 (HCN4) and Ciapin1, which are important proteins for MMWA treatment of insomnia. The iTRAQ-based proteomics data analyses presented here elucidate variations in the expression of proteins involved in the treatment of insomnia by MMWA.
Introduction
Surveys demonstrate that 31–56% of people in the world suffer from insomnia. Insomnia not only reduces immune function but also increases the risk of diabetes and hypertension. In recent years, insomnia has been mainly treated with drugs, and most drugs lead to resistance and dependence. Therefore, research on the nondrug treatment of insomnia is becoming urgent (Riemann et al. (Citation2020)).
Warm acupuncture therapy is a traditional external therapy method of Mongolian medicine that uses a special needle to give acupuncture at special acupoints of the human body combined with heat stimulation. Mongolian medical warm acupuncture (MMWA) has the advantages of high safety, strong practicality, and good development prospects (Wang and Li (Citation2017)).
MMWA therapy is an external treatment method combining acupuncture and warm moxibustion that can prevent diseases and enhance body resistance. It has been shown that warm acupuncture and moxibustion can improve sleep, soothe the nerves, and promote sedation. After warm acupuncture and moxibustion stimulated the acupoints (‘Dinghui’ acupoint, ‘Heyi’ acupoint, and ‘Xin’ acupoint) of P-chlorophenylalanine (PCPA)-induced insomnia rats, it was found that MMWA can regulate the expression of serotonin, norepinephrine, dopamine, gamma-amino butyric acid (GABA), glutamate, acetylcholine and other neurotransmitters (Li et al. (Citation2019), Bo et al. (Citation2016)).
To further study the molecular mechanism of MMWA and moxibustion in the treatment of insomnia, quantitative proteomics based on iTRAQ was used to determine proteins involved in the probable molecular mechanism of MMWA in the treatment of insomnia. The study showed that compared with the control group, the MMWA treatment group had a significant recovery. MMWA treatment of insomnia increases/decreases protein expression in the hypothalamus, including that of proline arginine (PREP), NMDA receptor synaptic nuclear signal and neuron migration factor (NSMF), transmembrane protein 41b (TMEM41b), and microtubule-associated protein 1B (MAP1B). The results of this study show that MMWA is effective in improving sleep by regulating protein expression in an experimental rat model and may have potential benefits in the treatment of insomnia patients without side effects. In a previous study, the tissue selected for analyzing the treatment of insomnia with MMWA was the hypothalamus (Xu et al. (Citation2020)). PCPA is commonly used to induce sleep deprivation models in rodents. PCPA acts on tryptophan hydroxylase by inhibiting enzyme activity and hindering 5-HT synthesis, resulting in the disappearance of sleep circadian rhythm, and induced insomnia (Shi et al. (Citation2018)). In this study, we selected the hippocampus as the proteomic tissue of PCPA insomnia rats.
The hippocampus is located between the thalamus and the brain’s medial temporal lobe. It is a part of the limbic system and is mainly responsible for the storage, conversion, and orientation of long-term memory. In addition, insomnia also affects the hippocampus, impeding the formation of long-term memory (Si et al. (Citation2017), Donato et al. (Citation2021)). In this study, the proteomics iTRAQ method was used to explore the proteomic changes induced by MMWA during insomnia treatment, and proteolytic peptide segments were labeled in hippocampal samples. In this study, we aimed to screen the potential neuronal differentially expressed proteins caused by MMWA treatment of insomnia, and the molecular mechanism of MMWA treatment of insomnia was discussed by bioinformatics system analysis.
Materials and methods
Materials
We purchased 30 Sprague Dawley rats aged 8–10 weeks and weighing 220 ± 10 g from the Experimental Animal Department of the Health Science Center of Peking University, and randomly divided them into three groups (n = 10/group): the control group (C), the insomnia model group (M), and the warm acupuncture group (W). Sample size was calculated using G * power software and the calculation parameters were set as follows: Test family: t tests; Statistical test: Means: Difference between two independent means (two groups); Type of power analysis: A priori: Computer required sample size give α, power, and effect size; Input Parameters: Tails – Two; Effect sized – 1.62352941; α err prob – 0.05; power – 0.9; Allocation ratio N2/N1 – 1. Sample size group 1 = 10, Sample size group 2 = 10. In this research, each group of animals was randomly divided according to the principle of equal probability. The experimental animals assigned to each group had the same gender, similar weight, and similar health status. The experimental groups were divided according to the random number table and randomized grouping table, and the personnel evaluating the results were not aware of the groupings. The animals were housed in an environment with the following conditions: 21–22 °C, 55–65% humidity, 12 h of light, 12 h of dark. Rats in each group were maintained in two cages and were regularly fed. Drinking water was freely provided, and they were raised adaptively for 7 days. No animals, experimental units or data points were excluded from the analysis. Animal experiments were completed at the animal experiment center of Inner Mongolia Medical University.
Rats in the M and W groups received intraperitoneal injections of 300 mg/kg PCPA (Sigma No: C6506, CA, USA) suspended in weakly alkaline saline (pH 7∼8) once daily between 8:30–9:00am for 2 consecutive days in order to establish insomnia rat models without any other stimuli.
After establishing the rat insomnia model, the W group received the acupuncture treatment. The acupuncture points Heyi, Dinghui, and Xin were selected (Supplementary Figure S1). Heyi and Xin are located in the centre of the superior fovea of the first and seventh thoracic vertebrae (upward and downward concavities, respectively). Dinghui is located at the centre of the parietal bone, at the intersection of the imaginary line connecting two earlobes and two eyebrow midlines (see Supplementary Figure S1). A stainless steel needle (0.35 mm × 20 mm from Inner Mongolia Yuanyang Traditional Chinese Medicine Co., Ltd.) was obliquely inserted and connected to the MWA needle device (MLY-I, developed by the College of Traditional Mongolia Medicine and Pharmacy of Inner Mongolia Medical University, patent no. ZL201120058078.0) set at a current intensity of 100 mA, temperature of 40 °C. The acupuncture depth of each point was 5 mm. The needle was retained for 15 min. The treatment was performed at 08:00 every morning and lasted for 7 days.
Rats in the C group underwent intraperitoneal injections of the same volume of a weakly alkaline saline solution only, once daily for 2 consecutive days.
After establishing each group of models, the rats were euthanized by cervical dislocation, the hippocampus of each rat was removed quickly and placed on ice, weighed, and preserved at −80 °C. In this research, we have followed the ARRIVE guidelines for the animal experiments.
Protein preparation
The hippocampus of all groups was harvested; SDT buffer was added to the sample and transferred into 2 mL centrifuge tubes prefilled with the appropriate amount of quartz sand and an additional 1/4 inch of ceramic beads. Next, the samples were homogenized in an MP tissue homogenizer (24 × 2, 6.0 M/S, 60 s, twice), ultrasonicated (80 W, 10 s ultrasound, 15 s interval, 10 cycles), and then incubated in boiling water for 15 min. After centrifugation at 14,000 × g for 40 min, the supernatants were filtered through a 0.22 µm filter. Protein concentrations were determined using the BCA method. Enzymatic digestion of 200 ug yielded a 100 ug peptide segment. The desalination column used for desalination had a load capacity of about 500 ug (Xu et al. (Citation2020)).
SDS–PAGE separation
For each sample, 20 μg of protein was added to 5× loading buffer and separated by 12.5% SDS–PAGE (constant current of 14 mA, 90 min).
Filter aided sample preparation (FASP) method
For each group, 200 μg of protein for each sample was incorporated into 30 μL of SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0). Next, the mixtures were boiled for 5 min and then cooled to room temperature. Next, 200 μL of UA buffer was added, mixed, transferred into 10 kDa ultrafiltration tubes and centrifuged at 14,000 × g for 15 min. The filtrate was discarded (this step was repeated once). Then, 100 μL of iodoacetamide (IAA) buffer (100 mM IAA in UA) was added, vortexed at 600 rpm for 1 min, and the mixture reacted for 30 min at room temperature under dark conditions. This was then centrifuged at 14,000 × g for 15 min. Then, 100 μL of UA buffer was added, and the sample was centrifuged at 14,000 × g for 15 min; the process was repeated twice. Then, 100 μL of dissolution medium diluted 10 times was added, and the sample was centrifuged at 14,000 × g for 15 min; this step was repeated twice. Next, 40 μL of trypsin buffer (4 μg trypsin in 40 μL dissolution buffer) was added, mixed by vortexing at 600 rpm for 1 min, and then incubated at 37 °C for 16–18 h. The sample column was transferred into a new collection tube and centrifuged at 14,000 × g for 15 min; then, the sample was eluted by adding 40 μL of 10 times diluted dissolution buffer, followed by centrifugation at 14,000 × g for 15 min. The peptides were desalinated using a C18 cartridge, and after lyophilization, they were resuspended in 40 μL of dissolution buffer. The peptide concentration was estimated by measuring the absorbance at OD280 (Xu et al. (Citation2020)).
iTRAQ labeling
A 100 μg peptide mixture of each sample was labeled using iTRAQ reagent according to the manufacturer’s instructions (Applied Biosystems). The iTRAQ experiment used 2D-LC-MS for peptide separation. The first dimensional chromatographic analysis mainly used SCX for preliminary separation of labeled peptide mixture samples, and the samples were separated into 10 components to reduce the complexity of peptide samples in each component. Then, these ten components were separately subjected to LC-MS to achieve better mass spectrometry identification results.
Peptide separation using strong cation exchange (SCX) chromatography
Acetonitrile (ACN) was used for peptide separation. Labeled peptides from each group were mixed and separated using the AKTAPurifier100. 10 mM KH2PO4 with 25% ACN (pH 3.0) was used as buffer A, and 10 mM KH2PO4, 500 mM KCL with 25% ACN (pH 3.0) was used as buffer B. The chromatographic column (Thermo scientific EASY column, 25 cm, ID75 μm, 1.9 μm) was equilibrated with buffer A, and samples were loaded into the column through a sample injector with a flow rate adjusted at 1 mL/min. The gradient was set as follows: linear gradient of 0–8% buffer B for 0–22 min; linear gradient of 8–52% buffer B for 22–47 min; linear gradient of 52–100% buffer B for 47–50 min; after 58 min, the buffer B concentration was set to 0%. During elution, the absorbance at 214 nm was recorded, and eluted solutions were collected every 1 min; a total of 30 eluted solutions were collected. After lyophilization, the samples were desalinated using a C18 cartridge (Xu et al. (Citation2020)).
Mass spectrometry analysis
HPLC
Samples were analyzed using Nano LC–MS/MS. Peptide mixtures were loaded with buffer A (0.1% formic acid) on a reversed-phase column and then eluted under a linear gradient of buffer B (84% ACN and 0.1% formic acid). The flow rate was adjusted at 300 nL/min (Xu et al. (Citation2020)).
LC–MS/MS analysis
After chromatography separation, samples were analyzed on a Q-Exactive mass spectrometer. Each sample required 60 min of analysis, and radical cations were used as fragment ions; the scan range was set at 300–1800 m/z. The resolution of the primary mass spectrometry was 70,000 at 200 m/z, and the AGC target was 3e6; the primary maximum IT was 10 ms, and the number of scan ranges was 1; dynamic exclusion was set at 40.0 s. The mass electron ratio of peptides and fractions were collected as follows: 10 fraction spectra were collected (MS2 scan) after each full scan; the MS2 activation type was HCD, and the isolation window was 2 m/z. The resolution of secondary mass spectrometry was 17,500 at 200 m/z; microscans: 1; the secondary maximum IT was 60 ms, normalized collision energy was 30 eV, and underfill was 0.1% (Xu et al. (Citation2020)).
GO analysis
NCBI BLAST + (NCBI last2.2.28+- win32. exe) was run, and the first 10 alignment sequences with an E value ≤ 1e-3 were saved for further analysis.
KEGG analysis
KAAS (KEGG Automatic Annotation Server) was used to analyze the KEGG pathway of the target protein cluster, classify the target protein sequence in KO terms, and then automatically extract the pathway information containing these target proteins according to KO classification.
Cluster analysis
Cluster3.0 software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) and Java Treeview (http://jtreeview.sourceforge.net) were used to calculate similarity.
Statistical analysis
The statistical software SPSS (version 22, SPSS, Chicago, Illinois, USA) was used to run Student’s t tests. Proteins with a > 1.2-fold change in expression (either up- or down-regulation) and p < 0.05 were considered to be significantly differentially expressed.
Results
LC–MS/MS
LC–MS/MS quality control showed that the molecular weight of the protein was in the range of 5 kDa to 100 kDa (Supplementary Figure S2), the length of the peptide was approximately 7–15 amino acids (Supplementary Figure S3), the distribution of mass error were close to zero, and the quality of the MS data met the requirements (Supplementary Figure S4). The whole experimental process is shown in Figure .
Figure 1 The entire experimental process. Experimental design for the quantitative proteomic analysis, the experiment is divided into three groups (control group, insomnia model group, and the warm acupuncture treated insomnia group). Protein extraction, trypsin digestion, and labeled with iTRAQ regents. The labeled peptides were separated by SCX chromatography and fractions were analyzed by reversed-phase LC-MS/MS, all data were analyzed by bioinformatics tools from different aspects (GO, KEGG).
Quantitative expression analysis of proteins
The differentially expressed proteins with a > 1.2-fold change in expression (either up- or down-regulation) and p < 0.05 were considered to be differentially expressed. In this study, a total of 6383 proteins were detected. Comparing M and C, 76 (45 up- and 31 down-regulated) differentially expressed proteins were observed. Comparing W and C, 149 (101 up- and 48 down-regulated) differentially expressed proteins were observed. Comparing W and M, 48 (26 up- and 22 down-regulated) differentially expressed proteins were observed. (Table )
Table 1 Quantitative expression analysis of proteins.
Cluster analysis
In the clustering results, red indicates up-regulation, and green indicates down-regulation. The results showed that the differentially expressed proteins changed significantly between M vs. C, W vs. C, W and M (Figures ), indicating the rationality of the differential expression mode of the selected target proteins.
Figure 2 Cluster analysis of differential level proteins between M vs. C. (Colors indicate the differential protein levels, which increase successively from green to red. Increased levels of proteins are indicated in red, and decreased levels are marked in green)
Figure 3 Cluster analysis of differential level proteins between W vs. C. (Colors indicate the differential protein levels, which increase successively from green to red. Increased levels of proteins are indicated in red, and decreased levels are marked in green)
Figure 4 Cluster analysis of differential level proteins between W vs. M. (Colors indicate the differential protein levels, which increase successively from green to red. Increased levels of proteins are indicated in red, and decreased levels are marked in green)
GO functional annotation and analysis
Compared to the C group, the molecular function analysis showed that the 76 differentially expressed proteins in the M group were mainly in enzyme regulator activity, catalytic activity, transporter activity, etc. Cell components of differentially expressed proteins were revealed in various kinds of locations, mainly in macromolecular complex, membrane, extracellular region, membrane-enclosed lumen, etc. Biological processes were allocated in single-organism process, metabolic process, and response to stimulus, signaling, localization, and the developmental process, etc. (Figure ).
Figure 5 GO analyses of protein functions in M vs. C. The GO functional annotations of 76 differentially expressed proteins in M vs. C. The 76 differentially expressed proteins were classified from three aspects: biological processes, molecular functions, and cellular components.
Compared to the C group, the molecular function analysis showed that 149 differentially expressed proteins in the W group were mainly in binding, catalytic activity, enzyme regulator activity, channel regulator activity, transporter activity, etc. Cell components of differentially expressed proteins were located mainly in macromolecular complex, membrane-enclosed lumen, organelle, membrane, extracellular, etc. Biological processes were allocated in single-organism process, metabolic process, the immune system process, localization, response to the stimulus, etc. (Figure ).
Figure 6 GO analyses of protein functions in W vs. C. The GO functional annotations of 149 differentially expressed proteins in W vs. C. The 149 differentially expressed proteins were classified from three aspects: biological processes, molecular functions, and cellular components.
For the W group compared to the M group, the result of molecular function analysis showed that 48 differentially expressed proteins were mainly in binding, catalytic activity, receptor activity, structural molecule activity, electron carrier activity, etc. Cell components of differentially expressed proteins were located mainly in membrane complex, organelle, macromolecular complex, extracellular, etc. Biological processes were allocated in single-organism process, metabolic process, response to stimulus, localization, signaling, etc. (Figure ).
Figure 7 GO analyses of protein functions in W vs. M. The GO functional annotations of 48 differentially expressed proteins in W vs. M. The 48 differentially expressed proteins were classified from three aspects: biological processes, molecular functions, and cellular components.
KEGG pathway analysis
To include cellular pathways involving W treatment with insomnia, we performed an enrichment analysis of KEGG pathways. The results show that for the M group compared with the C group, the differentially expressed proteins were involved in signaling pathways, including nitrogen metabolism (p value < 0.003653), hematopoietic cell lineage (p value < 0.009599), chemokine signaling pathway (p value < 0.016581), butanoate metabolism (p value< 0.01776), viral myocarditis (p value < 0.021545), Fc gamma R-mediated phagocytosis (p value < 0.028733) (Figure ). For the W group compared with C group, the differentially expressed proteins showed in malaria (p value < 0.002829), lysosome (p value < 0.012776), glycerophospholipid metabolism (p value< 0.025518), hematopoietic cell lineage (p value < 0.032669), linoleic acid metabolism (p value < 0.045604) (Figure ). For the W group compared with M group, there was enrichment in arachidonic acid metabolism (p value < 0.006005), linoleic acid metabolism (p value < 0.014929), thyroid hormone synthesis (p value < 0.027264), primary immunodeficiency (p value < 0.02942), glycosphingolipid biosynthesis-ganglioseries (p value < 0.02942), alpha-linolenic acid metabolism (p value < 0.036504) (Figure ).
Figure 8 KEGG analyses of protein functions in M vs. C. KEGG database pathway annotation was performed on 76 differentially expressed proteins in the insomnia model group with the control group.
Figure 9 KEGG analyses of protein functions in W vs. C. KEGG database pathway annotation was performed on 149 differentially expressed proteins in the warm acupuncture-treated insomnia group with the control group.
Figure 10 KEGG analyses of protein functions in W vs. M. KEGG database pathway annotation was performed on 48 differentially expressed proteins in the warm acupuncture-treated insomnia group with the insomnia model group.
Enrichment of neuronal proteins with differential levels in the warm acupuncture-treated group
The screening criteria were optimized. By comparing the differentially expressed proteins between each group, the results showed that the expression trends of 6 differentially expressed proteins between the W/M and M/C groups were opposite (Table ), suggesting that insomnia rats benefited from MMWA treatment. Insomnia may be treated by regulating protein expression. At the same time, the results showed that the comparison of differences between the W/M and W/C groups showed that the expression trend of 15 differentially expressed proteins in the W/C group was the same as that in the W/M group (Table ). Comparison results of differentially expressed proteins between groups suggest that warm acupuncture can treat insomnia by regulating the expression of these different proteins. It is suggested that differentially expressed proteins with MMWA treatment may be the key to the treatment of insomnia.
Table 2 Differentially expressed proteins between W/M vs. M/C groups.
Table 3 Differentially expressed proteins between W/M vs. W/C groups.
We performed an in-depth study of the screened target differentially expressed proteins. The expression of albumin (ALB) increased in W vs. M, and albumin contained sleep-promoting hormones, which could improve sleep. Through comparison of different target proteins between groups, it was found that Pro-MCH (PMCH), acetoacetyl-CoA synthetase (AACS), N-ethylmaleimide sensitive factor NSF (NSF), potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), and anamorsin (Ciapin1) are closely related to neuroregulation, suggesting that these differentially expressed proteins may be key to MMWA in the treatment of insomnia. These results provide novel clues and a theoretical basis for further study of the therapeutic mechanism of MMWA.
Discussion
Insomnia refers to the inability to sleep or maintain sleep, resulting in insufficient sleep and complex insomnia conditions. Insomnia patients generally have a prolonged latency of entering sleep, shortened sleep time, and increased physiological arousal in the process of falling asleep (Perez and Salas (Citation2020), Morin et al. (Citation2015), Brownlow et al. (Citation2020)).
MMWA can stimulate acupoints and transmit signals to the brain through nerves. Through the regulation of the cerebral cortex, it can relieve cerebral vasospasm, expand cerebral vessels, reduce cerebral vascular resistance, and improve cerebral circulation (Li et al. (Citation2018)). A previous study found that MMWA improves sleep and calms nerves and can significantly reduce the brain excitability of insomnia rats. It can promote the release of dopamine (DA) from the hypothalamus and prefrontal cortex of rats, reduce the content of glutamate, GABA, and acetylcholine in the hippocampus of insomnia rats, and reduce brain excitability (Tang et al. (Citation2019), Bo et al. (Citation2017)).
In this study, we used iTRAQ quantitative proteomics to identify proteins that are potential neural molecules involved in the treatment of insomnia by MMWA, and the molecular mechanism of MMWA treatment of insomnia was discussed by bioinformatics system analysis. At the same time, choosing the hippocampus to study the proteomics of insomnia rats is also one of the novel points of our study (Dogan (Citation2017), Wen et al. (Citation2020)).
The difference in protein expression caused by MMWA may be an essential element in the treatment of insomnia. In this study, we found that the components of differentially expressed proteins in the W group were mainly concentrated in macromolecular complexes, membrane sealed lumen, and cell membrane. The GO cell composition is consistent with the previous findings of hypothalamus proteomics in insomnia rats treated with MMWA (Xu et al. (Citation2020)).
Molecular functions mainly include catalytic activity, enzyme regulatory activity, channel regulatory activity, and electron carrier activity. MMWA mainly regulates biological processes such as metabolic processes, immune system processes, localization, and stimulation. However, according to GO analysis of the hypothalamic proteomics study of insomnia rats treated with MMWA, molecular functions were mainly enriched in signal transduction activation, antioxidant activity activation, transcription factor activation, and protein binding. Biological processes were mainly enriched in growth regulation, movement regulation and behavioral regulation (Xu et al. (Citation2020)). The proteomics of the hippocampus and the proteomics of the hypothalamus of insomnia rats treated with MMWA are different in the molecular function and biological process of GO in different tissues.
Through KEGG pathway enrichment analysis, it was found that the signaling pathways enriched by differentially expressed proteins after MMWA treatment were related to glycerol phospholipid metabolism, linoleic acid metabolism, arachidonic acid metabolism, and α-linolenic acid metabolism. Compared with the insomnia model group, it was found that there was a meaningful difference in the regulation of thyroid hormone synthesis after warm acupuncture treatment, and it was closely associated with ganglion glycosphingolipid biosynthesis. It was speculated that MMWA treatment of insomnia was closely related to the regulation of metabolism and hormone synthesis, which was the key to MMWA treatment of insomnia.
After in-depth screening of differentially expressed proteins between groups, ALB, PMCH, AACS, NSF, HCN4 and Ciapin1 were found to be involved in neuroregulation. These may be key influenced proteins in the process of MMWA.
It has been found that patients with chronic insomnia have abnormal ALB levels (Nie et al. (Citation2022)), and the duration of insomnia is closely related to ALB levels (Li and Guo (Citation2022)). ALB can maintain the body’s homeostasis (Ward et al. (Citation2022)), and can improve the sleep of rats (Obal et al. (Citation1998)). ALB is the carrier protein of hormones. ALB preparations may contain relatively high concentrations of these hormones, or endogenous humoral mechanisms stimulated by proteins (such as cholecystokinin or somatotropin axis) may mediate sleep enhancement after ALB. MMWA can promote an increase in ALB expression in insomnia rats, which may be a key factor in the treatment of insomnia (Sunnetcioglu et al. (Citation2016), Duger et al. (Citation2021)).
AACS is an enzyme that utilizes ketones to synthesize cholesterol and fatty acids. It is highly expressed in the brain and is a precious essential component of neuronal tissue. It was determined that AACS can regulate the growth of nerve axons in Neuro-2a cells (Hasegawa et al. (Citation2012), Hasegawa et al. (Citation2018)). In addition, AACS is regulated by SREBP-2 and participates in the normal development of neurons. AACS is found in the cytoplasm of the rat brain and may be important for brain development. Through the comparison of differentially expressed proteins between groups, it was found that after MMWA treatment, the expression of AACS in the hippocampus of insomnia rats increased, which may be related to the regulation of neuronal repair by MMWA by regulating the expression of AACS (Nomiyama et al. (Citation2019)).
NSF is an essential accessory protein of the neural transmission SNARE protein. NSF in rat synaptosomes has calcium-dependent phosphorylation. The phosphorylation of NSF occurs simultaneously with the release of neurotransmitters, which requires the inflow of external calcium to regulate neurotransmission (Santoro and Shah (Citation2020)). After MMWA treatment, the expression of NSF in the hippocampus of insomnia rats increased, which may be related to the regulation of neuronal repair by MMWA.
Hyperpolarization-activated (IH) current is important for dendritic integration, synaptic transmission, setting the membrane potential, and rhythmic action potential (AP) discharge of central nervous system neurons. Hyperpolarization-activated cyclicnucleotide-gated (HCN) channels form the basis for these currents. They are composed of homologous tetramers and heterotetramers of the HCN channel subunit (HCN1-4), which give channels different biophysical properties. HCN channel function is an important part of pain perception, learning and memory, sleep, and the pathogenesis of several nervous system diseases. The HCN4 subunit is expressed in several populations of the spinal cord and hippocampal interneurons, which are well known for expressing IH subthreshold currents and showing high-frequency AP discharge. HCN4 deficiency slowed thalamic and cortical oscillations during active wakefulness. The HCN4 channel is important for producing rhythmic intrathalamic oscillations and determining regular TC oscillation activity in an alarm state (Akimoto et al. (Citation2018), Cho et al. (Citation2009), Zobeiri et al. (Citation2019)). MMWA can increase HCN4 expression in insomnia rats, which may be a key factor in the treatment of insomnia.
Ciapin1 protein is widely expressed in the brain. In the ischemic animal model, the tat-ciapin1 protein was transported to the brain to protect against neuronal cell death in the hippocampal CA1 region induced by ischemic injury. Tat-ciapin1 protein has a protective effect on hippocampal neuronal cell injury induced by ischemic injury. At the same time, tat-ciapin1 protein may be a potential therapeutic agent for ischemia (Huang et al. (Citation2017), Wang et al. (Citation2016), Yeo et al. (Citation2021)). MMWA treatment may protect the brain from damage by increasing the expression of Ciapin1 protein in the hippocampus of insomnia rats.
PMCH melanin-concentrating hormone (MCH) is a peptidergic neuromodulator synthesized by neurons in the lateral hypothalamus and the adventitious zone. MCH neurons are distributed throughout the central nervous system and participate in various physiological functions. MCH is a cyclic neuropeptide that plays an important role in stimulating mammalian feeding behavior. MCH stimulates mammalian feeding and is a functional ‘super agonist’ (Dilsiz et al. (Citation2020), Bandaru et al. (Citation2020)). After MMWA treatment, the expression of MCH increases, which may be the key to regulating insomnia.
There are also limitations in this study that need to be presented. Whether the changes of differential proteins directly regulate insomnia or regulate signaling pathways is not clear. The key differential protein up/down regulations should be considered in future research.
Conclusion
Based on the iTRAQ proteomics analysis of the changes after the treatment of insomnia with MMWA, enrichment analyses for neuronal differentially expressed proteins revealed changes in ALB, PMCH, AACS, NSF, HCN4 and Ciapin1. Thus, regulating the function of neurons in insomnia is key to MMWA treatment of insomnia.
Authors’ contributions
Yanan Xu and Jianxun Wen contributed equally to this work. Xiulan Su and Gula A conceived and designed the experiments. Yanan Xu, Jianxun Wen performed the experiments and assessed the data. Wenyan Han performed language editing and data statistics. All authors read and approved the final manuscript and agreed to be accountable for all aspects of the research to ensure that the accuracy or integrity of any part of the work is appropriately investigated and resolved.
Supplemental Figures
Download MS Word (146.6 KB)Acknowledgment
We would like to show our great appreciation to Shanghai Applied Protein Technology Co., Ltd for the technical support of proteomics.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The data that support the findings of this study are openly available in ‘Zenodo’ at https://doi.org/10.5281/zenodo.8268067
Ethics approval and consent to participate
The animal experiments were approved by the Institutional Animal Care and Use Committee of Inner Mongolia Medical University (YKD2018096).
Additional information
Funding
References
- Akimoto M, VanSchouwen B, Melacini G. 2018. The structure of the apo cAMP-binding domain of HCN4 – A stepping stone toward understanding the cAMP-dependent modulation of the hyperpolarization-activated cyclic-nucleotide-gated ion channels. T FEBS J. 285:2182–2192. doi:10.1111/febs.14408.
- Bandaru SS, Khanday MA, Ibrahim N, Naganuma F, Vetrivelan R. 2020. Sleep-wake control by Melanin-Concentrating Hormone (MCH) neurons: a review of recent findings. CurrNeurolNeurosci Rep. 20:12–55.
- Bo A, Si L, Wang Y, Bao L, Yuan H. 2017. Mechanism of Mongolian medical warm acupuncture in treating insomnia by regulating miR-101a in rats with insomnia. ExpTher. 14:289–297.
- Bo A, Si L, Wang Y, Xiu L, Wu R, Li Y, et al. 2016. Clinical trial research on Mongolian medical warm acupuncture in treating insomnia. Evid Based Complement Alternat. 23:6190285–103.
- Brownlow JA, Miller KE, Gehrman PR. 2020. Insomnia and cognitive performance. Sleep Med Clin. 15:71–76. doi:10.1016/j.jsmc.2019.10.002.
- Cho HJ, Staikopoulos V, Ivanusic JJ, Jennings EA. 2009. Hyperpolarization-activated cyclic-nucleotide gated 4 (HCN4) proteins is expressed in a subset of rat dorsal root and trigeminal ganglion neurons. Cell Tissue Res. 338:171–177. doi:10.1007/s00441-009-0869-8.
- Dilsiz P, Aklan I, Sayar AN, Yavuz Y, Filiz G, Koksalar F, et al. 2020. Mch neuron activity is sufficient for reward and reinforces feeding. Neuroendocrinology. 110:258–270. doi:10.1159/000501234.
- Dogan A. 2017. Amyloidosis: insights from proteomics. Annu Rev Pathol. 12:277–304. doi:10.1146/annurev-pathol-052016-100200.
- Donato F, Alberini CM, Amso D, Dragoi G, Dranovsky A, Newcombe NS. 2021. The ontogeny of hippocampus-dependent memories. Neurosci. 41:920–926.
- Duger M, Seyhan EC, Gunluoglu MZ, Bolatkale M, Ozgul MA, Turan D, et al. 2021. Does ischemia-modified albumin level predict severity of obstructive sleep apnea? Sleep Breath. 25:65–73. doi:10.1007/s11325-020-02038-9.
- Hasegawa S, Imai M, Yamasaki M, Takahashi N, Fukui T. 2018. Transcriptional regulation of acetoacetyl-CoA synthetase by Sp1 in neuroblastomacells. Biochem Biophys Res Commun. 495:652–658. doi:10.1016/j.bbrc.2017.11.068.
- Hasegawa S, Kume H, Iinuma S, Yamasaki M, Takahashi N, Fukui T. 2012. Acetoacetyl-CoA synthetase is essential for normal neuronal development. Biochem Biophys Res Commun. 427:398–403. doi:10.1016/j.bbrc.2012.09.076.
- Huang Z, Su GF, Hu WJ, Bi XX, Zhang L, Wan G. 2017. The study on expression of CIAPIN1 interfering hepatocellular carcinoma cell proliferation and its mechanisms. Eur Rev Med Pharmacol Sci. 21:054–3060.
- Li J, Guo L. 2022. Association between sleep duration and albumin in US adults: a cross-sectional study of NHANES 2015–2018. BMC Public Health. 22(1):1102. doi:10.1186/s12889-022-13524-y.
- Li S, Bao L, Si L, Wang X, Bo A. 2018. Research on roles of Mongolian medical warm acupuncture in inhibiting p38 MAPK activation and apoptosis of nucleus pulposus cells. Evid Based Complement Alternat Med. 2018:6571320–39.
- Li X, Su B, Lian H, Bao M, Liang Y, et al. 2019. Effect of Mongolian warm acupuncture on the gene expression profile of rats with insomnia. Acupunct. 37:301–311. doi:10.1136/acupmed-2016-011243.
- Morin CM, Drake CL, Harvey AG, Krystal AD, Manber R, Riemann D, et al. 2015. Insomnia disorder. Nat Rev Dis Primers. 1:15026. doi:10.1038/nrdp.2015.26.
- Nie L, Pan XL, Zhang XB, Zhang S, Rao JX, Su ZF. 2022. Research on the correlation of immunity in patients with chronic insomnia. Front Psychiatry. 13:1034405. doi:10.3389/fpsyt.2022.1034405.
- Nomiyama R, Emoto M, Fukuda N, Matsui K, Kondo M, Sakane A, et al. 2019. Protein kinase C iota facilitates insulin-induced glucose transport by phosphorylation of soluble NSF attachment protein receptor regulator (SNARE) double C2 domain protein b. J Diabetes Investig. 10:591–601. doi:10.1111/jdi.12965.
- Obal FJ, Kapas L, Krueger JM. 1998. Albumin enhances sleep in the young rat. Physiol. 64:261–266.
- Perez MN, Salas R. 2020. Insomnia. Continuum (N Y). 26:1003–1015.
- Riemann D, Krone LB, Wulff K, Nissan C. 2020. Sleep, insomnia, and depression. Neuropsychopharmacol. 45:74–89.
- Santoro B, Shah MM. 2020. Hyperpolarization-activated cyclic nucleotide-gated channels as drug targets for neurological disorders. Annu Rev Pharmacol Toxicol. 60:109–131. doi:10.1146/annurev-pharmtox-010919-023356.
- Shi HY, Lu Y, Li YX, Tian FZ. 2018. Research progress of treatment with Chinese medicine for insomnia rats induced by PCPA. China Med Her. 15:33–36.
- Si Y, Chen X, Guo T, Wei W, Wang L, Zhang F, et al. 2017. Comprehensive 16S rDNA sequencing and LC-MS/MS-based metabolomics to investigate intestinal flora and metabolic profiles of the serum, hypothalamus and hippocampus in pChlorophenylalanine-induced insomnia rats treated with lilium brownie. Neurochem. 7:144–159.
- Sunnetcioglu A, Asker S, Alp HH, Gunbatar H. 2016. Increased asymmetric dimethyl arginine and ischemia-modified albumin levels in obstructive sleep apnea. Respir Care. 61:1038–1043.
- Tang L, You F, Hu X, Li YF. 2019. Electroacupuncture improves insomnia by down-regulating peripheral benzodiazepine receptor expression in hippocampus, and up-regulating 5-HT, 5-HIAA, TNF-alpha and IL-1beta contents in hypothalamus in insomnia rats. Zhen Ci Yan Jiu. 44:560–565.
- Wang J, Li Q, Wang C, Xiong Q, Lin Y, Sun Q, et al. 2016. Knock-down of CIAPIN1 sensitizes K562 chronic myeloid leukemia cells to imatinib by regulation of cell cycle and apoptosis-associated members via NF-kappaB and ERK5 signaling pathway. Biochem. 99:132–145.
- Wang L, Li G. 2017. Warm acupuncture for chronic atrophic gastritis with spleen-stomach deficiency cold. Zhongguo Zhen Jiu. 37:135–138.
- Ward ES, Gelinas D, Dreesen E, Van Santbergen J, Andersen JT, Silvestri NJ, Kiss JE, Sleep D, Rader DJ, Kastelein JJP, et al. 2022. Clinical significance of serum albumin and implications of FcRn inhibitor treatment in IgG-mediated autoimmune disorders. Front Immunol. 13:892534. doi:10.3389/fimmu.2022.892534.
- Wen B, Zeng WF, Liao Y, Shi Z, Savage SR, Jiang W, et al. 2020. Deep learning in proteomics. Proteomics. 20:e1900335–48.
- Xu Y, Li X, Man D, Su X. 2020. iTRAQ-based proteomics analysis on insomnia rats treated with Mongolian medical warm acupuncture. Biosci Rep. 40:BSR20191517. doi:10.1042/BSR20191517.
- Yeo HJ, Shin MJ, Kim DW, Kwon HY, Eum WS, Choi SY. 2021. Tat-CIAPIN1 protein prevents against cytokine-induced cytotoxicity in pancreatic RINm5F beta-cells. BMB Rep. 54:458–463. doi:10.5483/BMBRep.2021.54.9.040.
- Zobeiri M, Chaudhary R, Blaich A, Rottmann M, Herrmann S, Meuth P, et al. 2019. The hyperpolarization-activated HCN4 channel is important for proper maintenance of oscillatory activity in the Thalamocortical System. Cereb. 29:2291–2304. doi:10.1093/cercor/bhz047.