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Back to Journal »Neuropsychiatric Diseases and Treatment» Volume 17
Proteomic analysis reveals that Didang Tang prevents acute cerebral hemorrhage and stroke in rats by regulating S100a8, S100a9, Col1a1 and Col1a2
Authors: Feng Li, Li Ming, Ren Jie, Li Ya, Wang Qiang, Zhang Ping, Zhang Xu, Wang Teng, Li Ya
Published on November 10, 2021, the 2021 volume: 17 pages 3301-3314
DOI https://doi.org/10.2147/NDT.S331688
Single anonymous peer review
Editor who approved for publication: Dr. Ning Yuping
Lina Feng,1 Mingquan Li,2 Jixiang Ren,3 Yujuan Li,4 Qi Wang,5 Pengqi Zhang,1 Xinyue Zhang,1 Tianye Wang,5 Yunqiang Li1 1 School of Traditional Chinese Medicine, Changchun University of Traditional Chinese Medicine, Changchun City, Jilin Province, People's Republic of China 2 Department of Neurology, The Third Affiliated Clinical Hospital of Changchun University of Traditional Chinese Medicine, Changchun City, Jilin Province; 3 Department of Preclinical Preclinical Medicine, Affiliated Hospital of Changchun University of Traditional Chinese Medicine, Changchun City, Jilin Province; 4 Department of Ultrasound Diagnosis, The Third Affiliated Clinical Hospital of Changchun University of Chinese Medicine, Jilin Province Changchun City; 5 College of Integrated Traditional Chinese and Western Medicine, Changchun University of Traditional Chinese Medicine, Changchun City, Jilin Province Corresponding Author: Mingquan Li Tel 86-15543120222 Email [email protected] Purpose: This study aims to explore the neuroprotective mechanism of landmines and analyze Sprague Dawley through proteomics Dangtang (DDD) during stroke in rats with acute cerebral hemorrhage (AICH). Methods: 135 healthy Sprague Dawley rats were randomly divided into 5 groups: control group (n=27), model group (n=27), DDD low-dose group (n=27), DDD medium-dose group (n=27) , And DDD high dose (n = 27). AICH stroke in rats was induced by injecting autologous blood into the caudate nucleus. The modified neurological severity score (mNSS) is used to assess neurological deficits. Perform hematoxylin and eosin (HE) staining to observe the brain tissue of the lesion. When the blood-brain barrier is significantly damaged, the albumin concentration is evaluated, and the brain water content is evaluated for brain injury. For quantitative proteomics, proteins are extracted from the cerebral cortex. Use mass spectrometer-based targeted proteomics quantification to identify target proteins. Results: 7 days after exposure to DDD, the mNSS score, HE staining results, albumin concentration and brain water content of the high-dose group showed the most significant neuroprotective effects. In addition, quantitative proteomics analysis showed that compared with the control group, S100a8 and S100a9 were down-regulated by 0.614 times (p = 0.033702) and 0.506 times (p = 0.000024) in the high-dose group, respectively. Compared with the control group, Col1a1 and Col1a2 were up-regulated by 1.319 (p = 0.000184) and 1.348 (p = 0.014097) times in the high-dose group. These results were confirmed using mass spectrometer-based targeted proteomics quantification. Conclusion: The application of high-dose DDD in AICH stroke rats for 7 days shows the most significant neuroprotective effect. In terms of mechanism, this effect is mediated by down-regulation of S100a8 and S100a9 proteins and up-regulation of Col1a1 and Col1a2. Keywords: acute cerebral hemorrhage, stroke, S100a8, S100a9, Col1a1, Col1a2, Didang Decoction
Stroke is a cerebrovascular accident, a clinical event related to focal or diffuse brain dysfunction caused by acute cerebral circulatory disorders. Stroke is also the second leading cause of death from malignant tumors in urban and rural residents1. Stroke is divided into two categories according to its pathological nature: ischemic stroke and hemorrhagic stroke. Intracerebral hemorrhage (ICH) refers to hemorrhage in the brain parenchyma, which is characterized by rapid onset, deterioration of neurological function, and poor prognosis. 2 In China, 23.4% of stroke patients had acute cerebral hemorrhage (AICH), 46% of them died or were severely disabled within one year, 3 and 36% of survivors remained moderate to severely disabled at the time of discharge. 4 Many modifiable risk factors, including arterial hypertension, excessive consumption, low-density lipoprotein cholesterol, and low serum triglyceride levels can lead to AICH. 5 Conventional treatments for AICH include removing hematoma, reducing edema, and reducing intracranial pressure. However, unexpectedly, the effectiveness of the treatment was not satisfactory. 6 Therefore, it is necessary to develop new strategies for the treatment of ICH.
Traditional Chinese medicine is effective in treating stroke. 6Professor Ren Jixue has made outstanding contributions to traditional Chinese medicine encephalopathy, especially emergency department, and created a remarkable effect of "eliminating blood stasis" in the treatment of hemorrhagic stroke. In particular, Didang Decoction (DDD) is a classic traditional Chinese medicine prescription for acute hemorrhagic stroke, and a representative prescription for "eliminating blood stasis" in "On Febrile Diseases". Specifically, DDD promotes blood circulation, removes blood stasis, and effectively purifies the internal organs. DDD includes rhubarb (Latin name: Rheum palmatum L), peach kernels (Latin name: Prunus persica L. Batsch), leeches (Latin name: Whitmania pigra Whitman) and Gadfly (Latin name: Tabanus mandarinus Schiner). The use of DDD to treat AICH has a long history of proven effective clinical treatment. Our previous studies have shown that DDD significantly reduces brain water content and intracerebral hematoma volume in ICH rats by up-regulating the expression of brain-derived neurotrophic factor, tyrosine kinase B and vascular endothelial growth factor (VEGF). 7 Previous studies have also shown that Didang Decoction inhibits cell apoptosis mediated by endoplasmic reticulum stress induced by oxygen glucose deprivation and cerebral hemorrhage by blocking the GRP78-IRE1/PERK pathway. 8 We also found that the protective effect of Didang Decoction on AlCl 3 induced oxidative stress and apoptosis in PC12 cells is through the activation of SIRT1-mediated Akt/Nrf2/HO-1 pathway. 9 In addition, previous reports have shown that alcohol extract of leeches and water extract of rhubarb can reduce peripheral tissue inflammation and brain edema in ICH rats. 10,11 Peach water extract up-regulated VEGF and VEGF receptor 2 after ICH in a mouse model, and inhibited glucose deprivation in P to damage C12 cells. 12 However, its specific molecular mechanism for preventing acute cerebral hemorrhage and stroke in rats is still unclear. This study aims to explore the effect of DDD on AICH stroke rats and clarify its mechanism of action to guide its clinical use.
The experimental animal protocol was approved by the Animal Ethics Committee of Changchun University of Traditional Chinese Medicine (approval number: 20180008). All animal experiment procedures were performed in accordance with the guidelines of the National Institutes of Health on animal care and use. Animals are housed in animal dormitories approved by the International Association for the Evaluation and Certification of Laboratory Animal Care (AAALAC) in our hospital. The adult Sprague Dawley (SD) rats (180-220 g, 8-10 weeks old) from Changchun Yisi Experimental Animal Technology Co., Ltd. were studied. All animals are kept under the same conditions (room temperature 25° C and 12/12 hour light/dark cycle), and free access to food and water is allowed. Before inducing the AICH model, all rats were fed for 1 week.
According to the original record in the Dictionary of Traditional Chinese Medicine, DDT is composed of four traditional Chinese medicine components: rhubarb (Chinese name: rhubarb, Latin name: rhubarb, branch: Polygonaceae, batch number: 170916, use parts: roots and rhizomes), peach kernel (Chinese Name: Peach kernel, Latin name: Prunus persica L. Batsch, Family: Rosaceae, Lot number: 171011, Part of use: Seed), Leech (Chinese name: Water Zhi, Latin name: Whitmaniapigra Whitman, Family: Leech family, Lot number: 170824 , Use part: whole animal), Gadfly (Chinese name: Ding Chong, Latin name: Tabanus mandarinus Schiner, Family: Tetrigidae, batch number: 171015, Use part: whole animal). All dry ingredients were purchased from the Affiliated Hospital of Changchun University of Traditional Chinese Medicine, and were appraised by the Ministry of Medicine. First, the medicinal materials are chopped, mixed, soaked in distilled water for 30 minutes, and then decocted in distilled water at 100°C for 30 minutes. The above procedure was repeated 3 times, and the decoctions were combined to increase the concentration. The concentrated solution is freeze-dried under vacuum and ground into powder. According to the human and rat body surface area conversion equivalent dose ratio, dissolve the powder in distilled water to a final concentration of 0.625 g/mL (high dose), 0.3125 g/mL (medium dose) and 0.15625 g/mL (low dose) for later use.
As reported by our team, 8,9 we have established a method to detect the active ingredients in DDD by high performance liquid chromatography (HPLC, Agilent, Santa Clara, California, USA). The 18 main peaks of the DDD extract were identified using HPLC. The gallic acid, amygdalin, sennoside B, rhein 8-glucoside, sennoside A, emodin, chrysophanol, aloe-emodin and rhein in DDD were identified by comparing the retention time of high performance liquid chromatography , Good reproducibility.
135 healthy SD rats were randomly divided into 5 groups: control group, model group, DDD low-dose group, DDD medium-dose group, and DDD high-dose group. Each group uses 27 healthy SD rats. Each group is further divided into three subgroups, and each subgroup contains at least nine rats.
Model, low, medium and high dose groups were injected intraperitoneally with posterior pituitary injection 2U/kg, once a day for 14 days. After 14 days, the modified Nath method was used to replicate the AICH model. 8 Rats were injected intraperitoneally with 1% sodium pentobarbital and fixed in the supine position. Prepare the skin, separate the femoral artery, and ligate its distal end. In addition, 50 µL of femoral artery blood was extracted, and then the artery was ligated and surgically sutured. Subsequently, the rat was fixed in a stereotaxic instrument; the skin was prepared and routinely disinfected, the bregma was separated, the caudate nucleus was located, and 50 μL of autologous blood was slowly injected. After the operation, the scalp is sutured and penicillin powder is applied. The control group was the same except that autologous blood was injected into the caudate nucleus.
Judgment of model success: After the operation, the rats are fully awake, and the modified neurological severity score (mNSS) 13 is used to evaluate the neurological deficit of ICH model rats; mNSS score>6, hematoma formation can be seen in the perfused brain tissue, indicating the successful establishment of the model . If the symptoms of neurological dysfunction are too mild, non-existent, or too large, the rats with impaired consciousness, mobility difficulties, or death are discarded. After the model was successfully established, gavage was carried out immediately according to the respective dose concentration; the control group and the model group used the same amount of normal saline instead of DDD (1 mL/100 g body weight per group per day at the same time).
Rats in each group were sacrificed 1, 3, and 7 days after administration; then the cerebral cortex of the affected side was separated.
Two observers who were blind to the experimental design scored the animals independently and averaged the scores. These tests consisted of a modified neurological severity score (mNSS)13 and were repeated 3 times. mNSS nervous system score: 18 points of nervous system score are used (normal score 0; maximum defect score 18). For injury severity scores, points are scored for the inability to perform the test or lack of test reflex. Therefore, the higher the score, the more severe the injury.
After the paraffin-embedded sections are heated, the xylene gradient is used for dewaxing, and a gradient ethanol solution is added to remove the xylene. Place the slices in hematoxylin for 5-7 minutes and then wash with water for 1 minute. Add hydrochloric acid and ethanol (1%) for 10-30 seconds (depending on the tablet), then rinse under running water for 5-10 seconds. Place the slices in saturated lithium carbonate aqueous solution for 10-30 seconds, soak in tap water for 5-10 minutes, and soak in eosin solution for 20 minutes. Finally, wash the slices for 1 minute. Then use a gradient alcohol solution to dehydrate the sample, put it in fresh xylene, clarify it to achieve transparency, and seal it with a neutral gum. Observe pathological changes under light microscope.
The permeability of the blood-brain barrier is evaluated on the basis of the extravasation of the blood-brain barrier. 14 Due to the existence of the blood-brain barrier, the albumin concentration in the brain is generally very low, but once the blood-brain barrier is damaged, the albumin content in the brain tissue will increase significantly. Therefore, the change of albumin conversion rate can be used as an index to judge the degree of BBB damage. 15,16 Western blot analysis was used to detect the protein level of albumin in the brain tissue of rats in each group.
Western blot analysis: In short, collect, homogenize, and separately lyse brain samples around the hematoma in ice-cold RIPA lysis buffer (Beyotime, China). The sample was then centrifuged for 10 minutes (4 °C, 12,000 g). Collect the supernatant immediately and determine the protein concentration using the bicinchoninic acid (BCA) kit (Beyotime, China) according to the manufacturer's instructions. The protein sample (60 μg/lane) was separated by 10% or 12% SDS polyacrylamide gel and electrotransferred to the nitrocellulose filter membrane. The membrane was blocked with 5% skimmed milk at room temperature for 1 hour, and then incubated with the primary antibody overnight at 4°C. The membrane was then washed with TBST and incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) at room temperature for 2 hours. Use Enhanced Chemiluminescence (ECL) to visualize protein bands and use ImageJ software (National Institutes of Health) to determine relative protein amounts. The primary antibodies used include albumin (Sigma, USA) and β-tubulin (Sigma, USA) as loading controls. HRP-conjugated anti-IgG (Sigma, USA) was used as the secondary antibody.
As described in previous studies, rats were injected with 1% sodium pentobarbital when exposed to different concentrations of DDD and saline, and the intact brain tissue was immediately removed. The brain tissue is divided into two hemispheres along the midline. Use a cotton swab to gently remove the blood clot. After the filter paper absorbs the water on the brain surface, immediately weigh the brain tissue with an electronic analytical balance and record the wet weight (the weight is accurate to 0.1 mg). Then the brain tissue was dried in an electric constant temperature desiccator at 100±5°C for 72 hours, until the sample weight is composed of dry weight and calculated as follows: brain tissue water content = (wet weight-dry weight) / (wet weight) × 100 %.
Cut the prepared brain tissue (cerebral cortex on the affected side) into small pieces, add appropriate amount of PMSF RIPA, homogenize at low temperature, grade on ice, and centrifuge at high speed. The supernatant was then extracted and separated into 1.5 mL EP and stored at -20 °C.
For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 minutes at 56 °C and alkylated with 11 mM iodoacetamide for 15 minutes at room temperature in the dark. The protein sample was then diluted to a urea concentration of <2 M by adding 100 mM TEAB. Finally, trypsin was added at a trypsin to protein mass ratio of 1:50 for the first digestion overnight and a 1:100 trypsin to protein mass ratio for the second 4-hour digestion.
The digested peptides were desalted and vacuum dried using Strata X C18 SPE column (Phenomenex, USA). The peptide was dissolved in 0.5 M TEAB and labeled using the Tandem Mass Tag (TMT) kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. An Agilent 300 Extend C18 column (5μm, 4.6×250 mm) was used to fractionate the labeled peptides by high pH reversed-phase HPLC. First, a gradient of 8% to 32% acetonitrile (pH 9.0; Fisher Chemical, USA) was used to separate the peptides into 60 fractions within 60 minutes, and then they were combined into nine fractions and dried by vacuum centrifugation.
The peptide was dissolved in a liquid chromatography with mobile phase A (0.1% (v/v) formic acid in water) and separated using the EASY-NLC 1000 UPLC system. Mobile phase A: a solution containing 0.1% formic acid and 2% acetonitrile. Mobile phase B: a solution containing 0.1% formic acid and 90% acetonitrile. The liquid gradient settings are: 0-50 minutes, 7-16% B; 50-85 minutes, 16-30% B; 85-87 minutes, 30-80% B; 87-90 minutes, 80% B, flow rate maintaining At 400 nL/min. Peptides were analyzed using Orbitrap Fusion Lumos mass spectrometry. The scanning range and resolution of the primary mass spectrometer is 350-1550 m/z, 60,000. The scanning range and resolution of the secondary mass spectrometer is 100 m/z and 30,000. In the data acquisition mode, select the top ten peptide precursor ions with the highest signal intensity to enter the HCD collision cell for fragmentation, with a fragmentation energy of 32%, and proceed to the secondary mass spectrometry analysis in turn.
Use MaxQuant (v1.5.2.8) to search the MS mass spectrum data. The retrieval parameters are as follows: UniProt RAT is used to calculate the false alarm rate caused by random matching. A general pollutant database has been added to eliminate the influence of pollutant proteins on the identification results. The minimum length of the peptide is set to seven amino acid residues. The mass error tolerances of the primary precursor ions for the first and main searches are set to 20 and 5 PPM, respectively, while the mass error tolerance of the secondary fragment ions is 0.02. The quantitative method was set to TMT-10PLEX, and the FDR for protein identification and PSM identification was set to 1%.
Principal component analysis, relative standard deviation and Pearson correlation coefficient were used to evaluate whether the quantitative reproducibility of protein is statistically significant.
By setting 1.2 times and 1/1.2 times as the fold change threshold, determine the number of up-regulated and down-regulated proteins in each control group to determine the differential protein expression, and the statistical p value is <0.05.
Then classify the differential protein annotation from gene ontology (GO) as related to biological process (BP), molecular function (MF) or cell component (CC), and determine the subcellular location to annotate the biological role and protein location.
Based on GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) approach for functional enrichment analysis, p <0.05 (two-tailed Fisher's exact test) was considered statistically significant. Use the "heatmap.2" function in the "gplots" R package to perform one-way hierarchical clustering of differentially expressed proteins based on significant enrichment.
Select differentially expressed proteins for verification using RPM. RPM mass spectrometry analysis was performed using tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo, USA) coupled with UPLC online. Protein extraction, trypsin digestion, LC parameters, electrospray voltage, scanning range, and Orbitrap resolution were performed in the same way as the TMT experiment. The AGC of all MS is set to 20 M, and the MS/MS is set to automatic. The isolation window of MS/MS is set to 2.0 m/z. Use Skyline (V.3.6) to process the obtained MS data. After the quantitative information is normalized, relative quantitative analysis of the target peptide is performed.
All data are expressed as mean ± standard deviation (SD). Use Student's t-test and one-way analysis of variance (ANOVA) to analyze the data, and then use Scheffe's post hoc test. A one-way multivariate analysis of variance was performed to determine the significant differences in biochemical parameters between the control group and the sample group. At p <0.05, the difference is considered statistically significant.
On the first day, the four groups received autologous blood injection into the caudate nucleus to induce AICH and showed similar neurological deficits, while the animals in the control group had relatively no neurological deficits. Evaluation of mNSS at different time points showed that the neurological status of the model group and the DDD treatment group improved over time (mNSS decreased). The mNSS score of the high-dose group was significantly lower than that of the medium-dose group, low-dose group and model group on days 1, 3, and 7, and the difference was statistically significant (p <0.05), especially on day 7 (as shown in Figure 1. Show). Figure 1 The effect of DDD on the neurological results of AICH rats. *Compared with the model group, p <0.05; **Compared with the model group, p <0.01; ##Compared with the low-dose group, p <0.01; ▲▲Compared with the middle-dose group, p <0.01. Use Student's t-test and one-way analysis of variance (ANOVA) to analyze the data, and then use Scheffe's post hoc test.
Figure 1 The effect of DDD on the neurological results of AICH rats. *Compared with the model group, p <0.05; **Compared with the model group, p <0.01; ##Compared with the low-dose group, p <0.01; ▲▲Compared with the middle-dose group, p <0.01. Use Student's t-test and one-way analysis of variance (ANOVA) to analyze the data, and then use Scheffe's post hoc test.
In the animal control group, the non-necrotic cells in the cortex and medulla of normal rats were observed under a light microscope. In addition, these animals showed no blood injection into the brain, no inflammatory infiltration from the penumbra area, and had normal microscopic features. On the contrary, the pathological characteristics showed that the model group had obvious bleeding around the stove, infiltration of inflammatory cells, and edema around necrotic nerve cells. Compared with the AICH model group, the low, medium, and high dose groups showed more significant pathological features on the 3rd and 7th days, including hematoma formation, edema absorption, and anti-ischemic response of peripheral nerve cells in the ischemic area. area. In addition, the necrotic nerve cells around the lesion were significantly reduced. These results were especially obvious on the 7th day of the high-dose group (see Figure 2). Figure 2 HE staining. (A) Control group on day 3; (B) Model group on day 3; (C) Low dose group on day 3; (D) Medium dose group on day 3; (E) High dose on day 3 Dose group; (F) control group on day 7; (G) model group on day 7; (H) low-dose group on day 7; (I) medium-dose group on day 7; (J) 7th day High-dose group of days. (Box: obvious bleeding foci and bleeding points. Arrow: inflammatory cell infiltration. Straight line: typical neuronal morphology. Circle: vacuolar degeneration appears.).
Figure 2 HE staining. (A) Control group on day 3; (B) Model group on day 3; (C) Low dose group on day 3; (D) Medium dose group on day 3; (E) High dose on day 3 Dose group; (F) control group on day 7; (G) model group on day 7; (H) low-dose group on day 7; (I) medium-dose group on day 7; (J) 7th day High-dose group of days. (Box: obvious bleeding foci and bleeding points. Arrow: inflammatory cell infiltration. Straight line: typical neuronal morphology. Circle: vacuolar degeneration appears.).
We assessed the albumin level of each group, which is an important sign of BBB disruption. The brain tissue albumin level of the model group was significantly higher than that of the control group, while the low, medium, and high dose DDD treatment for 7 days significantly reduced the ICH-induced albumin level, and the high dose of DDD on the 7th day did not increase the albumin (such as Shown in Figure 3A-B). Regarding the brain water content, the model group increased significantly compared with the control group, while DDD treatment significantly impaired the brain water content. Compared with the cerebral hemorrhage model group, the brain water content of the low, medium and high dose groups on the 3rd and 7th day was significantly impaired, especially the high-dose DDD treatment on the 7th day compared with the control group (see Figure 3C). These results indicate , 7 days of high-dose DDD treatment can improve brain damage after ICH (including neurological results, BBB destruction and cerebral edema), and will not damage BBB. This restoration of BBB integrity may be an important factor in reducing brain edema after DDD treatment. Figure 3 Assessment of brain BBB destruction and brain water content. (A) Western blot analysis to check that the albumin level of Control, Model, DDD low-dose group on day 7, DDD medium-dose group on day 7, DDD high-dose group on day 7, and control group after exposure to DDD for 7 days; (B) The relative albumin level is calculated based on the optical density analysis. The average albumin level of the control group was standardized to 1.0; (C) brain water content recorded after exposure to DDD 1, 3, and 7 days (low dose, medium dose, high dose); *Compared with the control group, p <0.05;# Compared with the model group, p <0.05; ■ Compared with the low-dose group, p <0.05; ● Compared with the middle-dose group, p <0.05, ns is regarded as no significant difference, n = 9.
Figure 3 Assessment of brain BBB destruction and brain water content. (A) Western blot analysis to check that the albumin level of Control, Model, DDD low-dose group on day 7, DDD medium-dose group on day 7, DDD high-dose group on day 7, and control group after exposure to DDD for 7 days; (B) The relative albumin level is calculated based on the optical density analysis. The average albumin level of the control group was standardized to 1.0; (C) brain water content recorded after exposure to DDD 1, 3, and 7 days (low dose, medium dose, high dose); *Compared with the control group, p <0.05;# Compared with the model group, p <0.05; ■ Compared with the low-dose group, p <0.05; ● Compared with the middle-dose group, p <0.05, ns is regarded as no significant difference, n = 9.
Considering that the strongest effect was observed after 7 days of exposure to high-dose DDD, all subsequent experiments were performed under these conditions, unless otherwise stated. Differentially expressed proteins (high-dose group and model group) are classified according to their subcellular location (as shown in Figure 4A). Approximately 26.97% of differentially expressed proteins are present in the extracellular matrix, including collagen alpha-1 (I). Figure 4 Functional characterization and enrichment analysis of differentially expressed proteins. (A) Subcellular localization; (BD) Cluster analysis heat map of differentially expressed proteins based on GO (BP. MF. CC); (E and F) DDD based on KEGG enrichment expresses up-regulated and down-regulated proteins 7 days after exposure Cluster analysis heat map. Red indicates strong concentration (the darker the red, the stronger the concentration). Blue indicates weaker concentration (the lighter the blue, the weaker the concentration).
Figure 4 Functional characterization and enrichment analysis of differentially expressed proteins. (A) Subcellular localization; (BD) Cluster analysis heat map of differentially expressed proteins based on GO (BP. MF. CC); (E and F) DDD based on KEGG enrichment expresses up-regulated and down-regulated proteins 7 days after exposure Cluster analysis heat map. Red indicates strong concentration (the darker the red, the stronger the concentration). Blue indicates weaker concentration (the lighter the blue, the weaker the concentration).
Then GO classification (BP, MF, CC) and KEGG enrichment were performed for each group, and cluster analysis was performed to determine the correlation between protein function and differential expression. The result is visualized as a heat map. BP enrichment analysis showed that the up-regulated protein is mainly related to the sensory perception of mechanical stimulation. In contrast, the down-regulated protein was found to be largely related to bacterial response, inflammatory response to antigen stimulation, and leukocyte aggregation (as shown in Figure 4B). Regarding MF, down-regulated proteins are associated with Toll-like receptor 4 binding and antioxidant activity (as shown in Figure 4C). At the same time, for CC, centrosomes and nuclear chromosomes are mainly concentrated in down-regulated proteins (as shown in Figure 4D).
In order to further explore the DDD pathway during AICH on the 7th day, the KEGG pathway enrichment analysis was performed, and it was found that the up-regulated proteins were mainly enriched in the PI3k-Akt and AGE-RAGE signaling pathways (as shown in Figure 4E). At the same time, down-regulated proteins are mainly enriched in the IL-17 signaling pathway (as shown in Figure 4F).
These results indicate that the mechanism of DDD in the high-dose group during 7-day AICH may be related to the positive regulation of adaptive immune response, white blood cell aggregation and the regulation of inflammation-related signal pathways.
Venn diagrams were used to identify proteins that were significantly up-regulated and down-regulated before and 7 days after DDD administration (as shown in Figure 5). Among them, 23 proteins were up-regulated in the model group compared with the control group, and down-regulated in the analysis of the high-dose group and the model group. The 13 proteins in the model group and the control group were down-regulated, while the proteins in the high-dose group and the model group were up-regulated. Hierarchical clustering was performed on 3 repeated samples of the above 36 kinds of proteins to construct a heat map. The results indicate that these proteins may have similar functions and participate in certain related metabolic processes or signaling pathways (as shown in Figure 6). Figure 5 Distribution of differentially expressed proteins 7 days before and after exposure to DDD. Figure 6 Cluster analysis heat map of 36 differentially expressed proteins. Red represents up-regulated protein, and green represents down-regulated protein. The tree diagram at the top represents the cluster analysis results of different samples in different experimental groups. The dendrogram on the left represents the cluster analysis results of different genes from different samples.
Figure 5 Distribution of differentially expressed proteins 7 days before and after exposure to DDD.
Figure 6 Cluster analysis heat map of 36 differentially expressed proteins. Red represents up-regulated protein, and green represents down-regulated protein. The tree diagram at the top represents the cluster analysis results of different samples in different experimental groups. The dendrogram on the left represents the cluster analysis results of different genes from different samples.
Among the 36 proteins, S100a8 and S100a9 decreased by 0.614 and 0.506 times, respectively, after exposure to DDD for 7 days, while Col1a1 and Col1a2 increased by 1.319 and 1.348 times, respectively. See Table 1 for specific protein information. Table 1 The expression of target protein before and after DDD exposure for 7 days
Table 1 Target protein expression before and after exposure to DDD for 7 days
The S100 family is a group of low molecular weight modified binding proteins with similar structures and functions. S100a8 and S100a9 are two key members of the S100 family. They mainly play their biological functions when forming heterodimers. S100a8/a9.17 S100a8 and S100a9 play a role in a wide range of biological processes, such as apoptosis and immune inflammation Reaction and 18 Under abnormal conditions such as microbial infection and inflammation, activated immune cells secrete S100a8 and S100a9 to further activate the immune system and transmit signals to macrophages. 19 Macrophages can also stimulate inflammatory cells to release inflammatory factors, thereby directly Participate in acute and chronic inflammation. 20,21 Type I collagen is an important member of this family and a key structural component of the extracellular matrix, 22 as a result of subcellular localization. It is usually composed of type I collagen α1 chain (Col1a1) and type I collagen α2 chain (Col1a2). The expression and deposition of collagen can affect cell functions such as proliferation, adhesion, migration and invasion. 23,24 Col1a1 and Col1a2 affect vascular development, especially arterial development, venous vascular development and vascular maturation. And developmental abnormalities and even vascular malformations can lead to cerebral hemorrhage events. The functions of these four proteins are related to the results of bioinformatics analysis; therefore, the neuroprotective mechanism of DDD during AICH in SD rats may be mediated by regulating these proteins. Nevertheless, these results need further verification.
Quantify PRM based on peak area. In this experimental design, two or more unique peptides were used for each protein quantification. Due to sensitivity and other reasons, only one peptide has been identified for some proteins. PRM quantification of the target protein. The PRM results showed that four differentially expressed proteins, namely S100a8 (Figure 7A), S100a9 (Figure 7B and C), Col1a1 (Figure 7D) and Col1a2 (Figure 7E and F), showed the same trend as observed using quantitative proteomics Similar trends, and all results are statistically significant. Figure 7 PRM verification of the target protein. (A) S100a8 peptide corresponding ion peak area distribution; (B and C) S100a9 corresponding ion peak area distribution of different peptides; (D) Col1a1 peptide corresponding ion peak area distribution; (E and F) Col1a2 corresponding The area distribution of the different peptides of the ion peak.
Figure 7 PRM verification of the target protein. (A) S100a8 peptide corresponding ion peak area distribution; (B and C) S100a9 corresponding ion peak area distribution of different peptides; (D) Col1a1 peptide corresponding ion peak area distribution; (E and F) Col1a2 corresponding The area distribution of the different peptides of the ion peak.
DDD is a representative prescription for promoting blood circulation to remove blood stasis and purging the internal organs for more than 1800 years; it was first seen in "On Febrile Diseases". In fact, DDD composed of rhubarb, peach kernels, leeches, and gadfly has been shown to be effective for seemingly incurable diseases, including cerebral hemorrhage. Specifically, leeches are used to remove blood stasis without negatively affecting the newly formed blood because it only affects the blood phase and does not interfere with the qi. At the same time, the gadfly is responsible for blood extravasation and passes through the nine orifices. Considering that leaching is a monarch medicine, and gadfly is a minister medicine, the combination of the two can effectively solve the hemorrhagic disease. In addition, peach kernels are considered to be an auxiliary medicine that can reduce blood stagnation while helping to produce new blood. In the same way, rhubarb can also remove blood stasis and clear the stomach. Therefore, the combination of peach kernel and rhubarb can remove blood stasis and remove blood stasis. Combination of the four compounds can achieve the effect of promoting blood circulation, removing blood stasis and dredging collaterals. However, the exact relevant molecular mechanism is still unclear.
In this study, we investigated the neuroprotective mechanism of DDD on AICH rats. Hemorrhagic stroke is a particularly devastating disease, and its morbidity and mortality are higher than ischemic stroke. 25 In 2016, 13.7 million strokes occurred globally, 30% of which were hemorrhagic strokes. 26 According to the best practice recommendations for stroke in Canada: Management of Spontaneous Cerebral Hemorrhage, 7th Edition 2020 Update, 27 After diagnosis, one of the most important early treatment strategies is to limit the expansion of hematoma, which is the early deterioration of neurological function and poor clinical results Important determinants of 28 In the current study, compared with the AICH model group, the 3, 7-day low, medium, and high-dose groups had significant improvements in limiting hematoma expansion, edema absorption, and anti-ischemic response of peripheral nerve cells in the ischemic area. Moreover, the necrotic nerve cells around the lesion were significantly reduced. After 7 days of exposure to DDD, these results were particularly evident in the high-dose group. Using the mNSS score, we found that 7 days of high-dose DDD exposure has a powerful effect on improving neurological deficits and restoring BBB integrity. These results indicate that the application of DDD has had a positive impact on AICH.
Through bioinformatics analysis, these results indicate that the mechanism of DDD during 7-day AICH may be related to the active regulation of adaptive immune response, white blood cell aggregation and the regulation of inflammation-related signaling pathways. Next, use Venn diagrams to determine which proteins were significantly up-regulated and down-regulated 7 days before and 7 days after DDD exposure. Among them, 23 proteins were up-regulated in the model group and the control group, and down-regulated in the high-dose group and the model group. Compared with the control group, 13 proteins in the model group were down-regulated, while the high-dose group and the model group were up-regulated. In particular, compared with the control group, S100a8 and S100a9 were up-regulated by 3.316 and 4.957 times in the model group, respectively, while the high-dose group was down-regulated by 0.614 and 0.506 times compared with the model group. Col1a1 Col1a2 in the model group was down-regulated by 0.677 times and 0.598 times, respectively, compared with the control group, and the high-dose group was up-regulated by 1.319 and 1.348 times compared with the model group.
S100a8/a9 protein is a heterodimer composed of light chain S100a8 (molecular weight: 10,000) and heavy chain S100a9 (molecular weight: 14,000) proteins. S100a8 and S100a9 are mainly expressed in bone marrow cells. 17 According to structural studies, the S100A8/A9 protein complex contains two EF hand structures connected by a hinge region. 29 Infection-induced inflammation after bacterial infection can lead to the expression and secretion of neutrophils, macrophages and monocytes S100a8/a9.30 S100a8/a9 can promote inflammatory cell infiltration by releasing inflammatory cytokines, causing inflammatory damage Neutrophils accumulate at the site, and then release macrophages. S100a8/a9 produces a positive feedback effect, thereby amplifying the inflammatory response. 31 The early expression of S100 protein during infection-induced inflammation indicates that S100a8 and S100a9 trigger multiple inflammatory pathways mediated by TLR-4 or RAGE. 32 These considerations are combined with the results of the enrichment analysis of BP, MF and KEGG. Subsequently, the nuclear kappa B transcription signaling pathway is activated to stimulate downstream pro-inflammatory cytokines and inflammatory mediators. 33 Our research results showed that the protein levels of S100a8 and S100a9 in the high-dose DDD group were lower than those of the model group at 7 days, consistent with the PRM omics identification results, indicating that DDD may inhibit the infiltration of inflammatory cells, the release of inflammatory cytokines, and reduce the inflammatory response.
Type I collagen is the most abundant fibrous collagen in vertebrates and has been involved in the pathogenesis of aneurysms for a long time. Type I collagen is encoded by Col1a1 and Col1a2, and expresses α1(I) and α2(I). Polymorphisms of these genes have been confirmed to be related to vascular diseases. 34 The association analysis of primary intracerebral hemorrhage conducted by Ming et al. confirmed that the rs42524 polymorphism of the Col1a2 gene was significantly related to the occurrence of primary intracerebral hemorrhage. 35,36 Yoneyama et al. speculated that the Col1a2 gene polymorphism may affect the fragility and elasticity of the blood vessel wall and the interaction between other molecules, ultimately changing the strength of the blood vessel wall, and ultimately leading to ICH. 37 Col1a2 is the formation of an aneurysm. An important risk factor for the susceptibility of intracranial aneurysm has a strong impact in Asian populations. 38 Lindahl et al. showed that the genetic variation of Col1a2 is a risk factor for stroke and myocardial infarction. 39 Collagen type I related abnormalities are widespread in the scope of the disease, and several candidate genes have been identified40,41 Since type I collagen is widely distributed in the body, the influence of genetic variation may also affect the blood vessel wall and other tissues. It should also be noted that these patients sometimes develop aortic dissection,35 which is usually related to hypertension and arteriosclerosis. These two factors have always been considered risk factors for AICH. The protein expression of Col1a1 and Col1a2 in the DDD high-dose group was higher than that of the model group on the 7th day, which was also consistent with the results of PRM omics identification, indicating that the mechanism of DDD may be genetically related. Polymorphism and vascular development.
Based on current research, we propose that DDD may protect rats from AICH stroke by down-regulating S100a8 and S100a9 to inhibit inflammatory cell infiltration and release inflammatory cytokines. In addition, DDD may also affect vascular development by up-regulating Col1a1 and Col1a2. In addition, we plan to conduct follow-up experiments on modified proteomics to study the mechanism of DDD.
The application of high-dose DDD in AICH stroke rats for 7 days exerts neuroprotective effects by up-regulating Col1a1 Col1a2 and down-regulating S100a8 and S100a9 proteins.
All data generated or analyzed during this research period are included in this article.
This research protocol was reviewed and approved by the Animal Ethics Committee of Changchun University of Traditional Chinese Medicine (approval number: 20180008). All animal experiment procedures were performed in accordance with the guidelines of the National Institutes of Health on animal care and use. The animals are housed in the animal dormitories approved by the International Association for Laboratory Animal Care Evaluation and Certification (AAALAC) in our hospital, with a temperature control of 25°C and a light-dark cycle of 12 hours.
The author thanks Tian Yizhuang for his help in language editing, and Jingjie PTM Biological Laboratory (Hangzhou) Co., Ltd. for his help in mass spectrometry.
All authors have made significant contributions to the work of the report, whether in terms of concept, research design, execution, data acquisition, analysis and interpretation, or in all these areas; participating in drafting, revising or critically reviewing the article; final approval The version to be published; the journal to which the article is submitted has been agreed; and it has been agreed to be responsible for all aspects of the work.
This research was awarded the Jilin Province Science and Technology Development Plan Project [No. 20180101160JC].
The authors have no conflicts of interest to declare.
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