Searching for differentially expressed proteins in spinal cord injury based on the proteomics analysis

Journal Pre-proof Searching for differentially expressed proteins in spinal cord injury based on the proteomics analysis

Hai Ding, Jia Yu, Wenju Chang, Fendou Liu, Zhenxing He PII:

S0024-3205(19)31163-4

DOI:

https://doi.org/10.1016/j.lfs.2019.117235

Reference:

LFS 117235

To appear in:

Life Sciences

Received date:

29 September 2019

Revised date:

21 December 2019

Accepted date:

25 December 2019

Please cite this article as: H. Ding, J. Yu, W. Chang, et al., Searching for differentially expressed proteins in spinal cord injury based on the proteomics analysis, Life Sciences(2019), https://doi.org/10.1016/j.lfs.2019.117235

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© 2019 Published by Elsevier.

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Searching for differentially expressed proteins in spinal cord injury based on the proteomics analysis Hai Dinga, Jia Yub, Wenju Changa, Fendou Liua, Zhenxing Hea a

Department of Orthopedics, The First Affiliated Hospital of Bengbu Medical College,

Bengbu, Anhui 233004, People’s Republic of China Department of Ophthalmology, The First Affiliated Hospital of Bengbu Medical

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College, Bengbu, Anhui 233004, People’s Republic of China

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Corresponding author: Hai Ding

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Department of Orthopedics, The First Affiliated Hospital of Bengbu Medical College,

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No. 287 Changhuai Road, Bengbu, Anhui 233004, People’s Republic of China

Word count: 3453

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Figure count: 8

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Tel: 86-18655269280

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Email: [email protected]

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Abstract Aims: We aimed to identify potential differentially expressed proteins that play roles in the spinal cord injury. Materials and Methods: The mouse model of spinal cord injury was firstly built, followed by grip strength evaluation. Then, isobaric tags for relative and absolute

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quantization (iTRAQ) analysis was used to identify differentially expressed proteins at 1, 2, 3 and 8 weeks after spinal cord injury. Finally, analysis of spinal cord injury

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repair related differentially expressed proteins in the early and middle-late stage of

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injury was performed followed by the functional analysis.

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Key findings: The result of grip strength evaluation showed that the motor function of

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the forelimbs of the mouse was significantly impaired after spinal cord injury. In the

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iTRAQ analysis, a total of 29 common differentially expressed proteins (such as Hbb-bs, Hba, S100a6, Ca1, Apoa4, Hspb1, Hist1h1c, Hist1h1e, Hbb-b1, Apoa1 and

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S100a10) were obtained at 1, 2, 3 and 8 weeks after spinal cord injury. A total of 70 and 180 common differentially expressed proteins were identified in the early and middle-late stage of injury, respectively. PPAR signaling pathway (involved Apoa1) and VEGF signaling pathway (involved Hspb1) were identified in the middle-late stage of spinal cord injury repair. Significance: Identified differentially expressed proteins and related signaling pathways may be associated with spinal cord injury. Keywords: mouse model; spinal cord injury; Itraq; differentially expressed proteins; PPAR signaling pathway; VEGF signaling pathway 2

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Introduction Spinal cord injury (one of the major causes of disability) is divided into primary spinal cord injury and secondary spinal cord injury (1). The hallmark feature of spinal cord injury is the axonal disruption of the spinal cord. Neurological defect and disabilities caused by spinal cord injury affects not only the sensory and motor

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capabilities (such as paralysis, asthenia and spasticity) but also the physiological condition of patients (2-7). A systemic inflammatory response triggered by spinal cord

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injury leads to the high incidence of organ complications of patients (8-11). In

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addition, some clinical phenomenon including neurogenic pain, cardiovascular

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disease, depression, kidney dysfunction, liver damage and urinary tract infection

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developed by spinal cord injury affects life quality of patients.

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It is found that violence (with 26% of cases) and accidents (with 74% of cases) are the two factors of spinal cord injury (12-14). It is noted that severe spinal cord injury will

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lead to axonal loss, neuronal death, demyelination, neuronal connection disruption between brain and periphery, ultimately resulting in devastating loss of function (15, 16). Clinically, spinal cord injury is a disease with mortality occurring in patients prior to any primary hospital care, while patients treated at the hospital are also prone to mortality (17). Recently, the potential pathology mechanism of spinal cord injury remains unclear. Some researchers have investigated the molecular alterations pattern of spinal cord injury. Sengupta, MB et al found different protein expression profiles (such as VTDB vitamin D binding protein, Gc) in the cerebrospinal fluid of patients with a range of 3

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spinal cord injury severity (18). Chen, A and Kang, SK et al reported that various unique proteins were up-regulated in the lesion epicenter, such as peripherin, transferring, 14–3-3 zeta/delta, HSP90 and apolipoprotein A after spinal cord injury (19, 20). In addition, van Niekerk EA et al uncovered that several transcription factors (Jun, Smad, STAT, HNF, USF and SRF) were associated with gene expression

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changes after spinal cord injury (21). In order to deeply excavate differentially expressed molecules, we analyzed the differentially expressed proteins in week 1, 2, 3

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and 8 of spinal cord injuries in the mouse based on iTRAQ analysis. We also further

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identified spinal cord injury repair related proteins in the early and middle-late stages

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of spinal cord injury.

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Materials and Methods

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The mouse model of spinal cord injury

30 female mice (C57BL/6J, 20-25g) aged 8-10 weeks were supplied by Sibeifu

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company. These mice were given normal light cycles and normal diet. For further experiment, mice were randomly divided into two groups (15 mice per group): the cervical spinal cord injury group (injury group) and the sham operation group (control group). In the injury group, the spinal cord of mice was blunted contusion (a complete and bilateral injury). In the sham operation group, the mice received simple laminectomy. In each group, 3 mice were used for grip strength evaluation before spinal cord injury and 1 day, 3 days and 1-8 weeks after spinal cord injury, respectively. In addition, spinal cord tissues of another 12 mice (3 mice per week) were collected for iTRAQ analysis at 1, 2, 3 and 8 weeks after spinal cord injury. All 4

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experimental procedures were approved by The First Affiliated Hospital of Bengbu Medical College and raised carefully in accordance with National Institutes of Health on animal care and the ethical guidelines. Evaluation of grip strength in mouse It is well known that the motor function will be affected after spinal cord injury. It is

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noted that grip strength measurement is a reliable and sensitive method to evaluate the function of the forelimbs in the mouse with spinal cord injury. The baseline data of

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grip strength in the mouse was measured by grip force measuring instrument before

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the spinal cord injury and 1 day, 3 days and 1-8 weeks after spinal cord injury,

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respectively. Gently lifted mouse tail, induced the mouse to grasp the crossbar and

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quickly pulled the mouse off the crossbar backwardly. At this time, stress on the

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crossbar during pulling (the maximum grip force of the mouse’s two forelimbs for resisting pulling) was showed on the grip force measuring instrument. The grip

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strength of single left and right forelimb of the mouse was measured by the above methods when the side forelimb was wrapped with adhesive tape. The measurement result was recorded for 3 times and the mean value was taken. In order to ensure the accuracy of the data, the mouse should be given enough rest when tired during the measurement. iTRAQ The protein in injured spinal cord tissues was extracted followed by the concentration test. After protein enzymolysis, the iTRAQ reagent was applied to mark polypeptides. In the process, four isotope-coded tags are used to specifically label the amino group 5

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of the polypeptide. In order to test the efficiency and quantitative accuracy of labeling, 5μL was taken from each labeled sample (4 repeats) and mixed. The mixed sample and labeled samples were centrifuged and dried by vacuum freezing. The samples after being drained were freeze-preserved for use. The mixed samples were desalted by Ziptip and then detected by Orbitrap q-exactive plus to confirm that the labeling

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response was good. The strong cation exchange choematography was used for predissociation of balanced mixture of marked peptides. The liquid chromatography

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coupled with tandem mass spectrometry was utilized to analyze the differentially

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expressed proteins. Mascot software (version 2.5.1) was used to search the raw data

(sequence

total:

54448).

The

threshold

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uniprot-mus_54448_20190415.fasta

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after mass spectrometry. The database used in this study was UniProt database:

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(Abundance Ratio≥1.5 or Abundance Ratio≤0.667) of differentially expressed proteins was set as the criteria of statistical significance.

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Functional analysis of differentially expressed proteins To study the biological function of differentially expressed proteins at 1, 2, 3 and 8 weeks after spinal cord injury, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by using the online software GeneCodis3. The threshold of p value <0.05 was set as the criteria of statistical significance. Spinal cord repair related protein analysis in the early and middle-late stages of injury In order to find potential spinal cord repair related proteins and related signaling 6

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pathways in the early (week 1 and week 2) and middle-late (week 3 and week 8) stages of injury, we analyzed separately the common differentially expressed proteins in week 1 vs week 2 and week 3 vs week 8, respectively. In addition, we also performed the biological functional analysis of these common differentially expressed proteins.

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Validation in a published GEO dataset (GSE5296) The expression pattern of selected differentially expressed proteins was validated with

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GSE5296 dataset (involving 9 cases and 6 normal controls), which was downloaded

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from the GEO (https://www.ncbi.nlm.nih.gov/geo/).

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Statistical Analysis

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All statistical analyses were performed by GraphPad Prism (GraphPad Software, La

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Jolla, CA). Statistical significance was assessed by Student t-test. Statistical significance was ascribed to p value < 0.05. Graphs are presented as mean ± standard

Results

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error of the mean (SEM).

Grip strength evaluation

The result of grip strength evaluation of two forelimbs, single left forelimb and single right forelimb was shown in Figure 1A, 1B and 1C, respectively. In general, the grip strength of the mouse in the sham operation group was basically unchanged. The motor function of the forelimbs of the mouse was obviously impaired on the first 1 day of spinal cord injury. Furthermore, grip strength was significantly reduced compared with that before spinal cord injury. There was obvious grip change at week 7

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1 and week 2 of spinal cord injury. Grip strength is gradually improved from week 1 to week 4, indicating that the spinal cord injury was being repaired. After 4 weeks, the grip strength remained basically stable, but still lower than that of the control group. This suggested that the motor dysfunction of the mouse still existed. Grip strength of two forelimbs in the injury group was remarkably lower than that of the control group

Identification of differentially expressed proteins

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at each time point.

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The iTRAQ technology was used for differentially expressed protein identification in

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injured spinal cord tissues at 1, 2, 3 and 8 weeks after injury. Totally, 1089

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differentially expressed proteins were identified at 1, 2, 3 and 8 weeks after injury

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(Table 1). Among which, 29 commonly differentially expressed proteins were

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obtained at 1, 2, 3 and 8 weeks after injury, which was listed in Table 2. In addition, the plot of the relative protein abundance in each time point (1, 2, 3 and 8 weeks after

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injury) was shown in Figure 2. As showed in Figure 2, there was a significant difference between the injury group and the control group at 1 and 2 weeks. However, there was no significant difference between the injury group and the control group at 3 and 8 weeks, which may be due to better spine injury repair. The Venn diagram of differentially expressed proteins at week 1 vs control, week 2 vs control, week 3 vs control and week 8 vs control was shown in Figure 3. Figure 4 showed the heat map of 29 common differentially expressed proteins at 1, 2, 3 and 8 weeks after injury. Functional annotation of 29 common differentially expressed proteins According to the GO enrichment analysis, phospholipid efflux, cholesterol efflux and 8

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negative regulation of very-low-density lipoprotein particle remodeling were the most significantly enriched biological processes (Figure 5A); very-low-density lipoprotein particle, extracellular region and high-density lipoprotein particle were the most significantly enriched cellular components (Figure 5B); lipase inhibitor activity, high-density lipoprotein particle receptor binding and phosphatidylcholine-sterol

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O-acyltransferase activator activity were the most significantly enriched molecular functions (Figure 5C). In the KEGG enrichment analysis, only 5 signaling pathways

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were obtained. Among which, PPAR signaling pathway, vitamin digestion and

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absorption and fat digestion and absorption were the most enriched signaling

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pathways (Figure 5D).

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middle-late stages of injury

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Injury repair related differentially expressed proteins analysis in the early and

The grip strength result showed that there was obvious grip change at week 1 and

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week 2 of spinal cord injury. Therefore, it is needed to find spinal cord repair related differentially expressed proteins and related signaling pathways in the early (week 1 and week 2) and middle-late (week 3 and week 8) stages of injury. A total of 70 common differentially expressed proteins were identified in the early stage of injury. Additionally, a total of 180 common differentially expressed proteins were identified in the middle-late stage of injury. The Venn diagram of related differentially expressed proteins analysis in the early and middle-late stage of injury was shown in Figure 6A and Figure 6B, respectively. It is a pity that there was little result of biological functional analysis of differentially expressed proteins in the early stage of injury. 9

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After KEGG enrichment analysis of 180 differentially expressed proteins in the middle-late stage of injury, we found that PPAR signaling pathway (involved Apoa1) and VEGF signaling pathway (involved Hspb1) was two of enriched signaling pathways (Table 3). In addition, top 15 significantly enriched biological processes, cellular components, molecular functions and signaling pathways of these 180

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differentially expressed proteins were shown in Figure 7A, 7B, 7C and 7D,

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respectively. Validation in GSE5296

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Protein expression patterns of selected Hist1h1c, Hist1h1e, S100a10, S100a6, Apoa4,

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Apoa1 and Hspb1 were verified in GSE5296 dataset at week 1 and 4 after spine injury

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(Figure 8). The result showed that the protein expression levels of Hspb1, S100a10

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and S100a6 were significantly upregulated at both week 1 and 4 after spine injury; the protein expression levels of Hist1h1c, Hist1h1e, Apoa1 and Apoa4 were

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downregulated at week 4 after spine injury without significance, which was generally consistent with our iTRAQ analysis result. Discussion

Herein, we explored the grip strength of the mouse after spinal cord injury. Our result showed that the motor function of the mouse was significantly impaired after spinal cord injury. This suggested that spinal cord injury had an effect on the motor system function. After iTRAQ analysis, we obtained several important differentially expressed proteins such as Hist1h1c (histone cluster 1, H1c), Hist1h1e (histone cluster 1, H1e), Hbb-b1 (hemoglobin, beta adult major chain), Hbb-bs (hemoglobin, beta 10

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adult s chain), Hba (hemoglobin alpha chain complex), S100a10 (S100 calcium binding proteinA10), S100a6 (S100 calcium binding proteinA6), Ca1 (carbonic anhydrase 1) and Apoa4 (apolipoprotein A-IV). Hist1h1c plays a crucial role in multiple cellular processes, such as gene transcription, protein folding and apoptosis (22-25). The over expression of Hist1h1c was found in

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neural progenitor cells and early motor neurons (26). It is observed that Hist1h1c is up-regulated in the spinal cord of mouse (27). In addition, the expression of Hist1h1c

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is detected in patients with schizophrenia and severe Alzheimer’s disease (28, 29).

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Histone H1.4, encoded by Hist1h1e, mediates the formation of higher-order

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chromatin structures and regulates chromatin remodelling factor and the accessibility

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of regulatory protein (30, 31). It is found that Hist1h1e is over expression in neural

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progenitor cells and early motor neurons (26). In brain, Hist1h1e has been demonstrated as a crucial factor in micro vascular endothelial cells (32). It is reported

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that the expression of Hist1h1e is increased in the hippocampus and involved in nucleotide and nucleic acid metabolic process in the Alzheimer’s disease (33). Additionally, the haploinsufficiency of HIST1H1E protein and loss of function is associated with biological dysfunction in the brain (34). These reports demonstrated that Hist1h1c and Hist1h1e are key factors in neural progenitor cells and early motor neurons. In this study, we first found differentially expression of Hist1h1c and Hist1h1e in the injured spinal cord of mouse. In addition, the protein expression levels of both Hist1h1c and Hist1h1e were downregulated in the week 1 and 2, but upregulated in week 3. Our result suggested that Hist1h1c and Hist1h1e may play 11

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roles in the development of spinal cord injury repair. Hbb-b1, involved in the immune response, is differentially expressed in lumbar spinal cord (35). It is found that Hbb-b1 is up-regulated in spared nerve injury and associated with neuropathic pain (36). The up-regulation of Hbb-b1 is also reported in the peri-infarct area of experimental stroke (37). Hbb-bs is related to cell cycle and

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oxygen transport. The high expression of Hbb-bs is detected in the cortex and hippocampus of mouse (38). It has been suggested that Hbb-bs is down-regulated in

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ischemic stroke (39). Hba, a cytoskeleton-related gene, is differentially expressed in

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the spinal cord, glial cells and mesencephalic dopaminergic neurons (40, 41). It is

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demonstrated that Hba could increase neuronal survivability and functional

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performance after cerebral ischemia in gerbils (42). Kabanova S et al found that the

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expression of Hba was decreased in neurodegenerative diseases (such as Alzheimer’s disease and Parkinson’s disease). (43). It is noted that the expression of Hbb-bs and

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Hba was found in acute spinal cord contusion in rats (44). Herein, we found the expression changes of Hbb-b1, Hbb-bs and Hba in the injured spinal cord of the mouse. Our result indicated that Hbb-b1, Hbb-bs and Hba may be involved in the neural development of spinal cord injury. S100a10 (a member of the S100 protein family) is expressed in various cell types, such as astrocytes (45). It is reported that S100a10 releases neurotrophic factors and plays key roles in cell migration, which is contributed to neuroprotection (46-48). The expression of S100a10 is detected in neurons cells in distinct brain regions (49). It is noted that the role of S100a10 in human neuropsychiatric disorders has been reported 12

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in several clinical studies (50-53). S100a6, a glia-related gene, is involved in axonogenesis, myelination, synaptic transmission and neuronal differentiation, immune response and calcium signaling in spinal cord of mouse (54, 55). VanGuilder HD et al found that S100a6 is up-regulated with aging in the hippocampus and associated with neuroinflammation (56). In addition, the over expression of S100a6 is found in astrocytes of brainstem and spinal cord in patients with amyotrophic lateral

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sclerosis and Alzheimer’s disease (57, 58). These reports suggested that decreased

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expression of S100a6 could be beneficial. Herein, we found the differentially

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expression of S100a10 and S100a6 in the injured spine cord of mouse, which

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indicated that S100a10 and S100a6 could be related to neurogenesis in the process of

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spinal cord injury.

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The expression of Ca1 is detected in cerebrospinal fluid of patients with spinal cord injury (18). Ca1 plays an important role in effective tissue repair in acute spinal cord

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injury (59). Apoa4 has been detected in the central nervous system (60). Jiménez, CR et al found that Apoa4 was accumulated in the regenerating peripheral nerve and up-regulated in injured sciatic nerve (61). It is reported that Apoa4 is involved in neurological and psychiatric diseases, such as Alzheimer’s disease (62-65). In this study, we found the differentially expression of Ca1 and Apoa4 in the tissue of the injured spine cord of the mouse. Our result indicated that Ca1 and Apoa4 may be associated with spinal cord injury. In the middle-late stage of spinal cord injury, we found two important signaling pathways including proliferator-activated receptor (PPAR) and vascular endothelial 13

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growth factor (VEGF) that may be associated with spinal cord injury repair. It is worth mentioning that apolipoprotein A-1 (Apoa1) and heat shock protein 1 (Hspb1) were involved in the PPAR and VEGF signaling pathway, respectively. PPAR is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors (66). The PPAR subfamily is consisted of PPAR-α, PPAR-β and

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PPAR-γ (67). It is suggested that PPAR-α plays roles in controlling secondary inflammatory process of spinal cord injury (68). Moreover, genetic knockout of

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PPAR-α in mouse will exacerbate spinal cord damage (69). The expression of PPAR-γ

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is significantly down-regulated in spinal cord injury model rats (70). Additionally,

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PPAR-γ agonists could reduce marked neuron loss after spinal cord trauma (69). It is

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suggested that Apoa1 is a marker of neural degeneration and increased expression of

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Apoa1 in cerebrospinal fluid has been found in patients with Alzheimer’s disease (71).

(72).

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Significantly, Apoa1 has been identified as a potential biomarker of spinal cord injury

VEGF, a mitogen activated protein, is involved in the new blood vessels formation and cellular processes including growth, proliferation and survival. Although the expression of VEGF is lower in primary neurons, it plays a crucial role in the central nervous system undergoing spinal cord injury (73, 74). Choo, AM and Oh, JS et al found that VEGF could improve the survival of neural stem cell transplanted into injured spinal cords and enhances angiogenesis in spinal cord injury (75, 76). In addition, the delivery of VEGF into the spinal cord injury site will improve axonal regeneration after the injury (77). Hspb1 enhances neuronal survival and axonal 14

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regeneration (78). It is reported that the expression of Hspb1 decreased after spinal transection injury (79). It is showed that Hspb1 could alter restoration potential after nerve injury (80-82). Conclusions In a word, 9 differentially expressed proteins including Hist1h1c, Hist1h1e, Hbb-b1,

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Hbb-bs, Hba, S100a10, S100a6, Ca1 and Apoa4 may be involved in the development of spinal cord injury. In addition, PPAR and VEGF signaling pathways and involved

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differentially expressed proteins (Apoa1 and Hspb1) could be associated with spinal

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cord injury repair. However, there are limitations to our study. Firstly, the expression

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level of identified differentially expressed proteins is not verified in vitro, further

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validation is needed in human samples. Secondly, the deeper molecular mechanism of

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these differentially expressed proteins is not studied. Further tissue histopathologic analysis and cell experiment are needed.

None.

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Acknowledgments

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78. Siebert JR, Middleton FA, Stelzner DJ. Long descending cervical propriospinal neurons differ from thoracic propriospinal neurons in response to low thoracic spinal injury. Bmc Neuroscience. 2010;11(1):148. 79. Swieck K, Conta-Steencken A, Middleton FA, Siebert JR, Osterhout DJ. Effect of lesion proximity on the regenerative response of long descending propriospinal

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regeneration. Front Mol Neurosci. 2011;4:60.

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Figure legends Figure 1 The grip strength evaluation of mouse. Horizontal and vertical axis represents the injury time and grip strength, respectively. A: The grip strength evaluation of mouse two forelimbs; B: The grip strength evaluation of mouse single left forelimb; C: The grip strength evaluation of mouse

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single right forelimb. Figure 2 The plot of the relative protein abundance at 1, 2, 3 and 8 weeks after injury.

-p

*p<0.05; ***p<0.001.

ro

J: injury group; M: control group.

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Figure 3 The Venn diagram of differentially expressed proteins at week 1 vs control,

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week 2 vs control, week 3 vs control and week 8 vs control.

weeks after injury.

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Figure 4 The heat map of 29 common differentially expressed proteins at 1, 2, 3 and 8

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Diagram presents the result of a two-way hierarchical clustering of 29 common differentially expressed proteins and time points. The clustering is constructed using the complete-linkage method together with the Euclidean distance. Each row represents a differentially expressed protein and each column, a time point. Differentially expressed protein clustering tree is shown on the right. The colour scale illustrates the relative level of differentially expressed protein expression: red, below the reference channel; green, higher than the reference. Figure 5A Top 15 significantly enriched biological processes of 29 common differentially expressed proteins. 27

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Horizontal and vertical axis represents involved protein number and biological process, respectively. Figure 5B Top 15 significantly enrichment cellular components of 29 common differentially expressed proteins. Horizontal and vertical axis represents involved protein number and cellular

of

component, respectively. Figure 5C Top 15 significantly enrichment molecular functions of 29 common

ro

differentially expressed proteins.

-p

Horizontal and vertical axis represents involved protein number and molecular

re

function, respectively.

proteins.

na

lP

Figure 5D KEGG enrichment analysis of 29 common differentially expressed

Horizontal and vertical axis represents involved protein number and signaling

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pathway, respectively.

Figure 6A The Venn diagram of common differentially expressed proteins analysis in the early stage of injury (week 1 and week 2). Figure 6B The Venn diagram of common differentially expressed proteins analysis in the middle-late stage of injury (week 3 and week 8). Figure 7A Top 15 significantly enriched biological processes of 180 differentially expressed proteins in the middle-late stage of injury. Horizontal and vertical axis represents involved protein number and biological process, respectively. 28

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Figure 7B Top 15 significantly enrichment cellular components of 180 differentially expressed proteins in the middle-late stage of injury. Horizontal and vertical axis represents involved protein number and cellular component, respectively. Figure 7C Top 15 significantly enrichment molecular functions of 180 differentially

of

expressed proteins in the middle-late stage of injury. Horizontal and vertical axis represents involved protein number and molecular

ro

function, respectively.

-p

Figure 7D Top 15 significantly enrichment signaling pathways of 180 differentially

re

expressed proteins in the middle-late stage of injury.

lP

Horizontal and vertical axis represents involved protein number and signaling

na

pathway, respectively.

Figure 8 Validation of selected differentially expressed proteins in GSE5296.

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The x-axis shows injury (red color) and sham-injury (blue color) groups and y-axis shows the expression level.

A: Validation at week 1 after spine injury; B: Validation at week 4 after spine injury. *p<0.05; **p<0.05; ***p<0.001.

29

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Table 1 Differentially expressed proteins at 1, 2, 3 and 8 weeks after injury Compare group Week 1 vs Control

Week 2 vs Control

Regulated type

Number

up-regulated

387

down-regulated

49

up-regulated

6

down-regulated

122

up-regulated

213

436

128

295

of

Week 3 vs Control

Total

up-regulated

228

down-regulated

102

ro

82

330

Jo ur

na

lP

re

-p

Week 8 vs Control

down-regulated

30

Journal Pre-proof Table 2 Common differentially expressed proteins at 1, 2, 3 and 8 weeks after injury Wee Wee Wee Wee Week k1 Week k2 Week k3 Week k8 Accessio Gene 1 vs vs 2 vs vs 3 vs vs 8 vs vs n symb Contr Cont Contr Cont Contr Cont Contr Cont Number ol ol rol ol rol ol rol ol rol ratio up/d ratio up/d ratio up/d ratio up/d own own own own A8DUK Hbb-b 3.294 u 0.447 d 0.456 d 0.441 d 4 s 3641 p 5125 own 9157 own 3515 own Hba

3.386 9812 p

u

0.473 d 0288 own

APOA1

Apoa 1

2.428 3898 p

u

0.503 d 4778 own

0.493 d 1164 own

0.496 d 5462 own

PZP

Pzp

1.931 8727 p

u

0.535 d 8867 own

0.547 d 1469 own

0.532 d 1851 own

A0A0R4 J0I1

Serpi na3k

3.837 0565 p

u

0.578 d 3441 own

0.578 d 3441 own

0.578 d 3441 own

B3AT

Slc4a 1

2.445 2806 p

u

0.598 d 7394 own

0.594 d 6036 own

0.562 d 5292 own

EST1C

Ces1c

1.705 2698 p

u

0.562 d 5292 own

0.619 d 8538 own

0.615 d 5722 own

APOA4

Apoa 4

2.361 9853 p

u

0.562 d 5292 own

0.641 d 7129 own

0.543 d 3674 own

HSPB1

Hspb 1

5.314 7433 p

u

1.777 6854 p

CAH1

Ca1

2.329 4672 p

u

0.486 d 3275 own

0.543 d 3674 own

0.582 d 3668 own

VTDB

Gc

1.753 2114 p

u

0.655 d 1967 own

0.650 d 6709 own

0.611 d 3201 own

APOC1

Apoc 1

3.758 091 p

u

0.590 d 4963 own

0.637 d 2803 own

0.554 d 7847 own

ro

-p

re

lP

na

Jo ur

0.423 d 3727 own

of

HBA

31

u

4

u p

0.389 d 5823 own

3.160 1652 p

u

S100a 6

5.169 4113 p

u

1.790 0501 p

APOA2

Apoa 2

2.297 3967 p

u

0.456 d 9157 own

0.460 d 0938 own

0.493 d 1164 own

A1AT2

Serpi na1b

2.428 3898 p

u

0.641 d 7129 own

0.655 d 1967 own

0.646 d 1764 own

H14

Hist1 h1e

0.554 d 7847 own

0.602 d 9039 own

1.547 565 p

u

0.578 d 3441 own

Q91XL1

Lrg1

6.773 9625 p

u

0.460 d 0938 own

of

u

0.632 d 8783 own

0.453 d 7596 own

HBB1

Hbb-b 1

2.887 8584 p

u

0.444 d 4213 own

0.503 d 4778 own

A0A0R4 J1N3

Apoc 3

2.657 3716 p

u

0.550 d 9526 own

0.316 d 4391 own

HBBZ

Hbb-b h1

2.639 0158 p

0.411 d 7955 own

0.395 d 0207 own

0.486 d 3275 own

H12

Hist1 h1c

0.510 d 5061 own

0.650 d 6709 own

2.042 0243 p

u

0.598 d 7394 own

COBA1

Col11 a1

2.158 4565 p

u

0.554 d 7847 own

3.506 4229 p

u

4.626 7527 p

u

POSTN

Postn

2.361 9853 p

u

0.514 d 0569 own

1.505 2467 p

u

1.958 8406 p

u

S10AA

S100a 10

2.411 up 6157

1.658 up 6391

3.204 up 2795

2.928 1714 p

u

COMD8

Com md8

2.907 up 945

0.566 down 4419

0.503 down 4778

0.532 d 1851 own

COOA1

Col24 a1

1.515 up 7166

0.426 down 3174

9.126 up 1097

17.38 7758 p

u

A0A1Y7

Cpsf3

2.173 up

0.655 down

0.664 down

0.558

d

-p

0.514 d 0569 own

lP

Jo ur

na

u

5.098 2425 p

u

ro

S10A6

re

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32

6.453 1341 p

0.5

u

d own

Journal Pre-proof VLY3

4697

1967

3429

6436 own

Alox5 ap

3.784 up 2306

0.554 down 7847

2.027 up 919

1.765 406 p

MYPC2

Mybp c2

2.378 up 4142

0.411 down 7955

0.524 down 8583

0.521 d 2329 own

Jo ur

na

lP

re

-p

ro

of

AL5AP

33

u

Journal Pre-proof Table 3 KEGG analysis of 180 differentially expressed proteins in the middle-late stage of injury P valu e

Genes

Focal adhesion

13

9.35 E-12

Col11a1,Col6a3,Flnc,Itga5,Col6a1,Cav1,Pxn ,Actn1,Itga6,Col6a2,Flna,Col1a2,Col1a1

ECM-receptor interaction

9

2.37 E-10

Col11a1,Col6a3,Itga5,Col6a1,Itga6,Col6a2, Col1a2,Cd44,Col1a1

Lysosome

10

2.68 E-10

Ctsc,Hexb,Ctss,Ctsd,Ctsz,Ctsb,Lgmn,Scarb2, Tpp1,Lamp1

10

5.57 E-08

Wasf2,Itga5,Itgb2,Pxn,Iqgap1,Tmsb4x,Gsn, Msn,Actn1,Itga6

7

7.35 E-08

Col11a1,Col6a3,Col6a1,Col6a2,Col12a1,Col 1a2,Col1a1

5

3.35 E-06

C1qa,C1qc,Itgb2,Icam1,C1qb

6

1.47 E-05

Col11a1,Itgb2,Actn1,Col1a2,Hspb1,Col1a1

5

1.66 E-05

Wasf2,Itga5,Cav1,Pxn,Hcls1

6

1.70 E-05

Plcg2,Itgb2,Pxn,Msn,Actn1,Icam1

PPAR signaling pathway

5

3.21 E-05

Apoa2,Dbi,Fabp7,Apoc3,Apoa1

Fc gamma phagocytosis

5

5.10 E-05

Lyn,Wasf2,Plcg2,Marcks,Gsn

5

6.67 E-05

C1qa,Hist1h2bb,C1qc,Actn1,C1qb

4

7.57 E-05

Hexb,Npl,Cyb5r3,Uap1l1

actin

Protein digestion absorption

na

Amoebiasis

aureus

lP

Staphylococcus infection

and

Bacterial invasion epithelial cells Leukocyte migration

of

transendothelial

Systemic erythematosus

R-mediated

lupus

Amino sugar and nucleotide sugar metabolism

ro

of

-p

Regulation cytoskeleton

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Kegg: 0451 0 Kegg: 0451 2 Kegg: 0414 2 Kegg: 0481 0 Kegg: 0497 4 Kegg: 0515 0 Kegg: 0514 6 Kegg: 0510 0 Kegg: 0467 0 Kegg: 0332 0 Kegg: 0466 6 Kegg: 0532 2 Kegg: 0052 0

Term

re

Items

of

Co un t

34

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Antigen processing presentation

4

and

African trypanosomiasis

3

Complement and coagulation cascades

4

Cell adhesion (CAMs)

5

molecules

3

Hypertrophic cardiomyopathy (HCM)

4

Viral myocarditis

Icam1,Hbb-b2,Apoa1

C1qa,C1qc,Serpina1b,C1qb

Mpz,H2-D1,Itgb2,Itga6,Icam1

C1qa,C1qc,C1qb

re

lP 4

na

Hematopoietic cell lineage

H2-D1,Ctss,Ctsb,Lgmn

-p

Prion diseases

Itga5,Actn1,Itga6,Lmna

of

4

0.00 037 6 0.00 037 6 0.00 038 2 0.00 041 6 0.00 052 2 0.00 054 9 0.00 055 6 0.00 055 6 0.00 058 2 0.00 072 5 0.00 094 3 0.00 107 9 0.00 122 9 0.00 235 2 0.00 442

ro

Arrhythmogenic right ventricular cardiomyopathy (ARVC)

Jo ur

Kegg: 0541 2 Kegg: 0461 2 Kegg: 0514 3 Kegg: 0461 0 Kegg: 0451 4 Kegg: 0502 0 Kegg: 0541 0 Kegg: 0464 0 Kegg: 0541 6 Kegg: 0541 4 Kegg: 0414 5 Kegg: 0514 4 Kegg: 0515 2 Kegg: 0465 0 Kegg: 0452

4

Dilated cardiomyopathy

4

Phagosome

5

Malaria

3

Tuberculosis

5

Natural killer cell mediated cytotoxicity

4

Adherens junction

3

35

Itga5,Tpm4,Itga6,Lmna

Itga5,Itga6,Cd9,Cd44

H2-D1,Itgb2,Cav1,Icam1

Itga5,Tpm4,Itga6,Lmna

Itga5,H2-D1,Itgb2,Ctss,Lamp1

Itgb2,Icam1,Hbb-b2

Lsp1,Itgb2,Ctss,Ctsd,Lamp1

Plcg2,H2-D1,Itgb2,Icam1

Wasf2,Iqgap1,Actn1

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Vitamin digestion absorption Chagas disease trypanosomiasis)

3

and

(American

2

3

2

Proteasome

2

0.00 535 2 0.01 093 5 0.01 728 4 0.01 803 4

Plcg2,Pxn,Hspb1

Apoa4,Apoa1

C1qa,C1qc,C1qb

Apoa4,Apoa1

Psmb10,Psmb8

na

lP

re

-p

Fat digestion and absorption

0.00 496

of

VEGF signaling pathway

Jo ur

Kegg: 0437 0 Kegg: 0497 7 Kegg: 0514 2 Kegg: 0497 5 Kegg: 0305 0

5

ro

0

36

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5ab

Figure 5cd

Figure 6

Figure 7ab

Figure 7cd

Figure 8