Method for screening and determining biomarkers of ischemic stroke animal model of qi deficiency and blood stasis syndrome

Abstract

The application relates to a screening and determining method of an ischemic cerebral stroke animal model biomarker of qi deficiency and blood stasis syndrome, which integrates proteomics and transcriptomics technologies, systematically screens differentially expressed proteins and genes related to the syndrome, and on the basis, constructs various disease animal models, verifies candidate targets by adopting ELISA, simultaneously combines qPCR technology, multi-dimensionally confirms expression changes in the models, further draws ROC curves, calculates AUC values, sensitivity and specificity and other diagnostic performance indexes, evaluates diagnostic potential, and further knocks down and overexpresses differentially expressed genes, deeply excavates potential biomarkers with diagnostic values, and provides scientific bases for objective diagnosis of ischemic cerebral stroke qi deficiency and blood stasis syndrome and exploration of molecular mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of basic research technology, specifically relating to a method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome. Background Technology

[0002] "Syndrome" is the core of the theoretical system of Traditional Chinese Medicine (TCM), and syndrome differentiation and treatment are fundamental characteristics of TCM in treating diseases. However, the process of syndrome differentiation is deeply influenced by the physician's personal factors (such as the depth of understanding of TCM theory and clinical experience) and the patient's subjective feelings, making it difficult to achieve objectivity and quantification, thus leading to inconsistent diagnostic results. This problem directly affects the establishment of treatment methods, the selection of prescriptions and drugs, and the evaluation of clinical efficacy, thereby restricting the in-depth research and development of TCM. In basic TCM research, animal models are widely used to simulate the real process of diseases, especially in simulating the physiological and pathological states of "syndromes," where they are of great value. Therefore, using an animal model of ischemic stroke with qi deficiency and blood stasis syndrome has become an essential approach to exploring the essence of this syndrome.

[0003] This study, based on the traditional Chinese medicine theories of "exertion depletes qi," "hunger injures qi," and "qi and blood are mutually generative," successfully established a rat model of qi deficiency and blood stasis syndrome in ischemic stroke by combining fatigue, hunger, and suture occlusion. The model's symptoms and signs met the currently accepted criteria for identifying this syndrome model, indicating its effectiveness. However, the evaluation methods described above are still primarily subjective and lack highly specific and objective indicators. Whether the research results can scientifically reveal the essence of the syndrome and whether the established animal model is representative still depends on the objectivity and quantification of the diagnostic process. Therefore, establishing highly specific objective indicators to accurately assess the success of the qi deficiency and blood stasis syndrome model in ischemic stroke is crucial for advancing subsequent experimental research.

[0004] Traditional Chinese medicine believes that ischemic stroke originates from wind-evil invading the body, causing deficiency of the meridians and stagnation of vital energy, coupled with insufficient qi and blood, and imbalance of yin and yang, leading to damage to the internal organs and meridians. It is a condition of deficiency in the root and excess in the branch, and treatment should focus on tonifying qi and promoting blood circulation. Buyang Huanwu Decoction, first mentioned in *Yilin Gaicuo*, has a long history. Its characteristic formula is "not to expel blood stasis to promote blood circulation, but to tonify qi to promote blood circulation," pioneering a method of tonifying qi and promoting blood circulation that combines tonification and purgation. It is a representative formula for tonifying qi, promoting blood circulation, and unblocking the meridians, mainly used to treat diseases caused by qi deficiency and blood stasis leading to impaired blood flow.

[0005] During cerebral ischemia, the brain tissue suffers from impaired blood flow and insufficient oxygen supply, leading to a lack of substances and energy required for metabolism. Mitochondrial dysfunction is a crucial link in the pathological process of Qi deficiency and blood stasis syndrome in ischemic stroke. Furthermore, Qi deficiency and blood stasis syndrome is related to coagulation and anticoagulation dysfunction and microthrombus formation in modern medicine. Clinically, hemorheology and coagulation function are often used as objective evaluation indicators for Qi deficiency and blood stasis syndrome. Therefore, this experiment, in addition to TCM syndrome indicators, also tested hemorheology, coagulation function, and mitochondrial morphology indicators, providing objective quantitative standards for evaluating Qi deficiency and blood stasis syndrome in ischemic stroke.

[0006] With the development of high-throughput sequencing technology, omics technologies are playing an increasingly important role in disease research. Proteomics studies the proteome, investigating how different proteins interact and their functions within organisms. Transcriptomics studies gene expression at the RNA level—the sum of all RNA transcribed from a living cell—and is a crucial tool for studying cell phenotype and function. As research into the relationship between Traditional Chinese Medicine (TCM) syndromes and proteomics and transcriptomics deepens, it has become clear that the manifestations of TCM syndromes are essentially the expression levels of specific functional proteomes and their RNA in an individual, and the differences between different TCM syndromes are essentially due to variations in the expression levels of their functional proteomes and transcriptomes.

[0007] Syndrome differentiation is based on three elements: macroscopic characteristic combination, microscopic indicator characteristic combination, and prescription counter-syndrome. Given the current lack of in-depth research on biomarkers for Qi deficiency and blood stasis syndrome in ischemic stroke, this study recorded the symptoms, signs, and other TCM syndrome indicators of rats with Qi deficiency and blood stasis syndrome in ischemic stroke. These were combined with objective indicators such as rat hemorheology, coagulation function, and mitochondrial morphology to evaluate the success of the rat model of ischemic stroke. Furthermore, proteomics and transcriptomics techniques were used to reveal the molecular characteristics of Qi deficiency and blood stasis syndrome in ischemic stroke to identify biomarkers. Finally, the classic Qi-tonifying and blood-activating formula, Buyang Huanwu Decoction, was used as a control drug for intervention to improve Qi deficiency and blood stasis syndrome in ischemic stroke. Summary of the Invention

[0008] To address the problems existing in the prior art, the purpose of this invention is to solve the lack of objective diagnostic criteria for Qi deficiency and blood stasis syndrome in ischemic stroke. To achieve the above objective, this invention employs the following technical solution: A method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome, characterized by the following steps: 1) Construct an animal model of ischemic stroke with qi deficiency and blood stasis syndrome; 2) Based on the model animals and control animals obtained in step 1), perform proteomics and transcriptomics analyses respectively to screen for differentially expressed proteins and genes; 3) Perform joint analysis on the differentially expressed proteins and genes obtained in step 2), and take the intersection molecules that differ at both the protein level and the transcription level as candidate biomarkers; 4) Verify the expression of the candidate biomarkers identified in step 3) in animal models and evaluate their diagnostic efficacy to identify biomarkers for ischemic stroke with qi deficiency and blood stasis syndrome.

[0009] Furthermore, the animal model in step 1) is the SD rat model.

[0010] Further, the construction steps of step 1) are as follows: first, a model of qi deficiency and blood stasis syndrome is established through hunger and fatigue intervention, and then an ischemic stroke model is established through middle cerebral artery occlusion.

[0011] Further, in step 3), the joint analysis specifically involves taking the intersection of the gene corresponding to the differentially expressed protein and the differentially expressed gene to obtain common targets that show significant differences at both the protein and transcriptional levels.

[0012] Further, in step 3), the identified candidate biomarkers include at least one of Mx1, Irf8, Cfd, Cd22, Pf4, Golm1, Pycard, Dnah12, Fgl2, Lcp2, Shc1, Sparc, Ifngr1, Tinagl1, Btk, Lrg1, Adh6, Rps19, Fapp5, Tlr3, Casp6, Fyb1, Nt5c, Fn1, and Usp4.

[0013] Further, in step 4), the verification includes verifying the expression of the candidate biomarker at the protein level using enzyme-linked immunosorbent assay (ELISA), and / or verifying its expression at the mRNA level using quantitative polymerase chain reaction (PCR).

[0014] Further, in step 4), the assessment of diagnostic efficacy includes plotting the receiver operating characteristic curve and calculating the area under the curve.

[0015] Furthermore, the candidate biomarkers are Sparc, Ifngr1, and Fapp5.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1) Based on the TCM theories of “exertion depletes qi”, “hunger injures qi”, and “qi and blood are mutually generating”, a rat model of qi deficiency and blood stasis syndrome in ischemic stroke was constructed using fatigue, hunger and suture occlusion method. Proteomics and transcriptomics technologies were used to provide a scientific basis for identifying qi deficiency and blood stasis syndrome in ischemic stroke. 2) This invention is based on proteomics and transcriptomics. After repeated verification and screening, the selected test proteins can reflect the pathological nature of qi deficiency and blood stasis syndrome in ischemic stroke. 3) Enzyme-linked immunosorbent assay (ELISA) can specifically detect macromolecular antigens and specific antibodies in rat brain tissue, and has outstanding advantages such as convenient operation, high sensitivity, good reproducibility, and easy standardization of solid-phase carriers. This invention combines omics technology with ELISA technology to screen for marker proteins associated with the Qi deficiency and blood stasis syndrome in ischemic stroke, successfully identifying marker proteins related to this syndrome. This indicator can serve as an important basis for judging this syndrome type in animal models, thereby achieving efficient, simple, and objective assessment of the Qi deficiency and blood stasis syndrome status in ischemic stroke, and is suitable for rapid screening of animal models that meet specific criteria. 4) This invention can further promote the convenience and objectivity of TCM diagnosis and facilitate the scientific evaluation of animal models in basic experiments. Attached Figure Description

[0017] Figure 1 Changes in body weight (g) of rats in each group (compared to the Control group), ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05); Figure 2 Forelimb grip strength (g) of rats in each group (compared with the Control group, ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05); Figure 3 The ratio of cerebral infarction area to total area and neurological function scores of rats in each group (compared with the Control group) ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05); Figure 4 Tongue appearance of rats in each group; Figure 5 Blood rheological parameters of rats in each group (compared with the Control group) ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05); Figure 6 Coagulation function indices (s) of rats in each group (compared with the Control group, ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05); Figure 7 Observation of mitochondrial morphology in rats of each group; Figure 8 Differentially expressed proteins and gene analysis; Figure 9 Venn diagram of the intersection genes of the proteome and transcriptome; Figure 10 Differential protein expression levels in the proteome; Figure 11 differentially expressed gene levels in the transcriptome (compared to the control group), ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05); Figure 12 Expression levels of Sparc, Ifngr1, and Fapp5 proteins ( ** P <0.01, * P <0.05); Figure 13 Sparc, Ifngr1, and Fap5 mRNA expression levels ( ** P <0.01, * P <0.05); Figure 14 ROC curves for Sparc, Ifngr1, and Fapp5; Figure 15 Comparison of forelimb grip strength (g) among different groups of rats (compared to the Coontrol group) ## P <0.01, # P <0.05; compared with the Model group, ** P <0.01, * P <0.05); Figure 16 Comparison of hemorheological parameters of rats in each group (compared with the control group), ## P <0.01, # P <0.05; compared with the Model group, ** P <0.01, * P <0.05); Figure 17 Comparison of coagulation function indices among rat groups (s) (compared with the Control group, ## P <0.01, # P <0.05; compared with the Model group, ** P <0.01, * P <0.05); Figure 18 Comparison of neurological function scores among different groups of rats (compared with the Control group) ## P <0.01, # P <0.05; compared with the Model group, ** P <0.01, * P <0.05); Figure 19 Comparison of forelimb grip strength among different groups of rats ( ** P <0.01, * P <0.05); Figure 20 Comparison of neurological function scores among different groups of rats ( ** P <0.01, * P <0.05); Figure 21Comparison of hemorheology indexes in each group of rats ( ** P <0.01, * P <0.05); Figure 22 Comparison of coagulation function indexes in each group of rats ( ** P <0.01, * P <0.05). Specific implementation manners

[0018] The following further describes the present invention in conjunction with specific embodiments for better understanding the technical solution.

[0019] Example 1: Establishment and evaluation of a model of rats with qi deficiency and blood stasis syndrome of ischemic stroke 1 Experimental animals 48 SPF-grade SD male rats (3 - 7 weeks old, body weight 220 ± 20 g), purchased from Shanghai Slack Experimental Animal Co., Ltd., license number: SYXK (Zhe) 2021 - 0012, and raised in the Animal Experiment Center of Zhejiang Chinese Medical University. They were adaptively raised for 1 week before the experiment.

[0020] 2 Experimental methods 2.1 Preparation of Buyang Huanwu Granules Astragalus membranaceus 60 g, Angelica sinensis 6 g, Paeonia lactiflora 5 g, Pheretima aspergillum (prepared with wine) 3 g, Ligusticum chuanxiong 3 g, Carthamus tinctorius 3 g, Prunus persica (prepared with boiling water) 3 g. Preparation method: The above herbs were purchased and identified by the Pharmacy Department of the First Affiliated Hospital of Zhejiang Chinese Medical University (Zhejiang Provincial Hospital of Traditional Chinese Medicine), prepared according to the ratio, soaked, decocted, decocted again, the liquid was taken, the two decoction liquids were mixed, concentrated, spray-dried, packaged, and granule preparations were obtained.

[0021] 2.2 Animal grouping and administration After 7 days of adaptive feeding, the SD rats were randomly divided into 4 groups, with 12 rats in each group, namely the Control group, the Model group, the BYHW-L group, and the BYHW-H group. According to the equivalent dose calculated based on the body surface area between humans and rats, the administration doses of Buyang Huanwu Decoction were selected as the low dose (BYHW-L group) 1.74 g / Kg / 24 h and the high dose (BYHW-H group) 6.94 g / Kg / 24 h. The Control group and the Model group were given the same volume of solvent.

[0022] 2.3 Construction of a model of rats with qi deficiency and blood stasis syndrome of ischemic stroke 2.3.1 Construction of the qi deficiency and blood stasis syndrome model (1) Simulation of starvation factor The average daily feed requirement for rats is 186-374 g / kg, and feed should be provided at 60% of the normal diet. Animals should be weighed, and two-thirds of the daily feed should be given at 8:00 AM, with the remaining one-third given at 8:00 PM.

[0023] (2) Fatigue Rats were placed in batches into a circular swimming pool with an inner diameter of 150 cm and a water depth of 40 cm after being loaded with 4% ± 0.5% of their body weight. The water temperature was controlled at (20 ± 1) °C. They were forced to swim continuously until exhaustion and then retrieved. The signs of exhaustion were incoordination in swimming movements, water submerging the nose, and being unable to surface for more than 10 seconds after sinking. Adaptive swimming training was conducted for 3 days, followed by exhaustive swimming once a day for 14 days.

[0024] 2.3.2 Construction of a Qi Deficiency and Blood Stasis Syndrome Model for Ischemic Stroke The day after the Qi deficiency and blood stasis syndrome model was established, the MCAO model was prepared using the modified Zea-Longa suture occlusion method. After weighing, the rats were anesthetized, fixed, and prepared. A midline incision was made in the neck, and the right common carotid artery, external carotid artery, and internal carotid artery were bluntly dissected. The distal end of the external carotid artery was ligated, and a loose knot was tied 2–3 mm from the common carotid artery. The common carotid artery and internal carotid artery were clamped. A small incision was made between the ligation site and the loose knot in the external carotid artery, and the suture was inserted and pushed into the internal carotid artery approximately 18 mm. The incision was sutured, disinfected, and reperfusion was performed 60 minutes later.

[0025] 2.4 Evaluation of a rat model of ischemic stroke with qi deficiency and blood stasis syndrome 2.4.1 Evaluation of the Qi Deficiency and Blood Stasis Syndrome Model (1) Body weight, grip strength, tongue appearance (2) Hemorheology (3) Coagulation function (4) Mitochondrial morphology 2.4.2 Evaluation of Ischemic Stroke Models (1) Zea-Longa neurological function score (2) Stroke infarct area / total area Example 2: Screening of differentially expressed proteins and genes 1. Screening for differentially expressed proteins, the specific steps are as follows: a) Sample preparation ① Protein extraction: Take 30uL of sample, add 600uL of LIPA lysis buffer, mix well, sonicate in an ice water bath for 20min, centrifuge at 12000rpm for 10min, and transfer the supernatant to a new EP tube.

[0026] ② BCA quantification: Add 200 μL of BCA working solution to each well of a 96-well plate, with 7 standard spots and 1 blank. Add 20 μL of sample (diluted accordingly) or standard protein (BSA). Shake at 37°C for 30 min, and measure the absorbance at 562 nm. Fit a standard curve based on the standard protein and calculate the protein concentration of the corresponding sample.

[0027] ③ Reduction & Alkylation & Proteolytic Digestion: Take 40 μg of protein, add 5 times the volume of pre-cooled acetone, and precipitate at -40℃ for 4 h. Centrifuge at 12000 rpm and 4℃ for 20 min, discard the supernatant, and add 30 μL of reagent to dissolve the precipitate. Add 20 μL of reagent A, mix well, and incubate at 1000 rpm and 95℃ for 5 min. Cool the sample to room temperature, add 15 μL of reagent B, mix well, and digest at 1000 rpm and 37℃ for 2 h. After digestion, add 55 μL of reagent C, mix well, and terminate the digestion reaction. Add all the sample to the desalting column, centrifuge at 700g for 1 min, and repeat once. Add 100 μL of reagent D to the desalting column, centrifuge at 700g for 1 min, and repeat once. Add 100 μL of reagent E to the desalting column, centrifuge at 700g for 1 min, and repeat once. Place the desalting column in a new centrifuge tube, add 100 μL of reagent F to the desalting column, centrifuge at 700g for 1 min, and repeat once. The final sample volume is 200 μL. Concentrate the sample using a vacuum refrigerated centrifuge concentrator. After reconstitution, add the sample to an iRT for analysis.

[0028] b) NanoLCMS / MS detection ① For each sample, approximately 500 ng of total peptides was separated using a nano-UPLC liquid chromatography system and then data was acquired using a mass spectrometer equipped with a nanoliter ion source. The mobile phase consisted of an 80% acetonitrile-water-formic acid system, where mobile phase A was a 0.1% formic acid aqueous solution and mobile phase B was a 0.1% formic acid-80% acetonitrile solution. After equilibration of the column with 96% of phase A, the sample was directly loaded onto the column using an autosampler and then subjected to gradient separation at a gradient duration of 8 min.

[0029] ② The mass spectrometry analysis used the data-independent acquisition (DIA) mode, with a total analysis time of 8 minutes, and positive ion detection mode.

[0030] ③ The normalized collision energy (NCE) is 25%. The full scan range is 150-2000 m / z.

[0031] c) Library search and identification, and protein quantification 2. Screening for differentially expressed genes, the specific steps are as follows: ①The RNA in the total sample was isolated and purified using Trizol.

[0032] ② The quantity and purity of total RNA were quality controlled, and the integrity of the RNA was tested.

[0033] ③ Use magnetic beads to specifically capture the polyA-containing mRNA through two rounds of purification.

[0034] ④ The captured mRNA was fragmented under high temperature conditions.

[0035] ⑤ Fragmented RNA is synthesized into cDNA by reverse transcriptase.

[0036] ⑥ Perform double-strand synthesis, convert these DNA and RNA complex double strands into DNA double strands, fill in the ends of the double-stranded DNA to blunt ends, and add an A base to each end so that it can be linked to the adapter with a T base at the end. Then use magnetic beads to screen and purify the fragments by size.

[0037] ⑦ Digest the two strands to form a strand-specific library.

[0038] ⑧ Perform paired-end sequencing according to standard operating procedures.

[0039] Example 3: Validation at the protein level using ELISA technology. Includes the following steps: a) Preparation of protein from rat brain tissue in each group Rat brain tissue was collected, homogenized with pre-cooled RIPA lysis buffer, and the supernatant was collected by centrifugation.

[0040] b) Protein detection in rat brain tissue of each group Prepare the standards and washing buffer according to the ELISA kit instructions. After adding the samples, seal the plate and incubate at 37°C. Discard the buffer and wash. Add the enzyme-labeled antibody and substrate sequentially for color development, and then add the stop solution. Measure the absorbance at 450 nm using a microplate reader and calculate the contents of Sparc, Ifngr1, and Fapp5 in brain tissue according to the standard curve.

[0041] Example 4: Validation at the gene level using qPCR technology Includes the following steps: a) RNA extraction: 50 mg of rat ischemic cortex was taken and RNA was extracted from the brain tissue using the Trizol method.

[0042] b) RNA concentration detection: RNA concentration was determined using an ultra-micro spectrophotometer.

[0043] c) Reverse transcription: Based on the sample concentration calculations, the reaction system was prepared and cDNA was synthesized according to the instructions of the Zhenbai reverse transcription reagent. The names and amounts of each component are shown in Table 1, and the reaction conditions are shown in Table 2. The reverse transcription reaction was carried out on ice, and the reaction process is shown in Table 3.

[0044] Table 1

[0045] Table 2

[0046] d) Amplification: Add cDNA, target gene primers, ddH2O, and 2X SYBR Green ProTaq HS Premix to each of the eight-tube sets. Perform the amplification reaction according to the instructions of the Zhenbai dedicated qPCR reaction kit. Use the β-actin internal reference gene blank as a control. Results are obtained using 2... -△△Ct Calculations were performed. The names and amounts of each component are shown in Table 3, the reaction conditions are shown in Table 4, and the primer sequences used for qPCR analysis are shown in Table 5.

[0047] Table 3

[0048] Table 4

[0049] Table 5

[0050] Example 5: Through repeated verification of the above experiments, the ELISA and qPCR detection results of rat brain tissue proteins in each group were compared and analyzed. Differential genes with expression trends consistent with those screened by proteomics and transcriptomics were selected as biomarkers for qi deficiency and blood stasis syndrome in ischemic stroke.

[0051] The results of the above experiment are as follows: 1. Evaluation of animal models To evaluate the effects of Buyang Huanwu Decoction on a rat model of Qi deficiency and blood stasis, this study systematically observed changes in macroscopic characteristics and microscopic indicators. The results showed that, compared with the Control group, the Model group rats exhibited significantly inhibited weight gain and forelimb grip strength. Figure 1 , Figure 2 Table 6 P <0.01), and the appearance of typical blood stasis manifestations such as a purplish-dark tongue, indicating that the model of qi deficiency and blood stasis syndrome was successfully constructed. Figure 4 Regarding cerebral ischemia-related injury, the infarct size / total brain area ratio in the Model group rats was significantly increased (47.90%±1.36%), and the neurological deficit scores were significantly elevated (1.83±0.37). Figure 3 Table 7P The result was <0.01, further confirming that the model exhibits significant pathological changes associated with ischemic stroke.

[0052] After intervention with Buyang Huanwu Decoction, the above-mentioned symptoms of qi deficiency and blood stasis were significantly improved. The animals' weight and gripping strength recovered significantly. Figure 1 , Figure 2 (Table 6) The tongue appearance is approaching normal. Figure 4 Meanwhile, the neurological function score gradually decreased with increasing dosage. Figure 3 Table 7 P <0.01%. Buyang Huanwu Decoction effectively reduced the area of ​​cerebral infarction ( Figure 3 Table 7 P <0.01, and reversed the model-induced abnormalities in blood rheology and coagulation function, manifested as a decrease in whole blood high-shear blood relative index, whole blood low-shear blood relative index, erythrocyte aggregation index, and shear rate. Figure 5 Table 8), and the increase in APTT and PT coagulation parameters ( Figure 6 Table 9 P <0.05. Regarding ultrastructure, Buyang Huanwu Decoction treatment also significantly improved pathological changes in the Model group, such as mitochondrial cristae breakage, membrane dissolution, and rough endoplasmic reticulum lesions. Figure 7 ).

[0053] In summary, Buyang Huanwu Decoction has multifaceted therapeutic effects on rats with qi deficiency and blood stasis syndrome complicated by ischemic stroke. This formula not only alleviates qi deficiency and blood stasis by promoting weight gain, restoring forelimb grip strength, and improving tongue appearance, but also significantly reduces the area of ​​cerebral infarction, promotes the recovery of neurological function, and effectively regulates blood rheology, coagulation function, and mitochondrial structure. Furthermore, the overall therapeutic effect is dose-dependent.

[0054] Table 6. Changes in body weight (g) of rats in each group

[0055] Compared to the Control group, ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05.

[0056] Table 7. Cerebral infarction area / total area and neurological function scores of rats in each group.

[0057] Compared to the Control group, ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05.

[0058] Table 8. Blood rheological parameters of rats in each group

[0059] Compared to the Control group, ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05.

[0060] Table 9. Coagulation function indicators (s) of rats in each group

[0061] Compared to the Control group, ## P <0.01, # P <0.05; compared with the Model group ** P <0.01, * P <0.05.

[0062] 2. Results of differential protein screening Through systematic proteomics analysis of the Control, Model, and BYHW groups, we screened out a total of 406 significantly differentially expressed proteins. Figure 8 These extensive protein expression perturbations reveal the complex molecular mechanism of qi deficiency and blood stasis syndrome in ischemic stroke at the systemic level, and also provide a wealth of candidate targets for in-depth analysis of the pharmacological effects of Buyang Huanwu Decoction.

[0063] 3. Results of differential gene screening At the transcriptome level, we identified a total of 1032 differentially expressed genes that were significantly associated with disease status and drug intervention. Figure 8This gene set not only helps to elucidate the core molecular drivers of disease development, but also provides important clues for discovering potential biomarkers for Buyang Huanwu Decoction.

[0064] 4. Joint analysis and screening results 4.1 Venn diagram of the intersection of proteome and transcriptome genes To accurately pinpoint key targets, we integrated a differential molecular analysis of the proteome and transcriptome. By taking the intersection of Venn diagrams, we successfully identified 25 common targets that showed significant differences at both the protein and transcriptional levels, including Mx1, Irf8, Cfd, Cd22, Pf4, Golm1, Pycard, Dnah12, Fgl2, Lcp2, Shc1, Sparc, Ifngr1, Tinagl1, Btk, Lrg1, Adh6, Rps19, Fapp5, Tlr3, Casp6, Fyb1, Nt5c, Fn1, and Usp4. Figure 9 This highly consistent group of molecules is very likely the core hub driving the pathological process of Qi deficiency and blood stasis syndrome and being regulated by Buyang Huanwu Decoction.

[0065] 4.2 Display of differentially expressed genes Further screening of differentially expressed genes with high expression levels in rat brain tissue cortex revealed that Sparc, Ifngr1, and Fapp5 were ultimately identified as biomarkers for qi deficiency and blood stasis syndrome in ischemic stroke. Figure 10-11 This represents the changes in differentially expressed gene levels between the proteome and transcriptome. Example

[0066] By constructing different disease models, we used ELISA and qPCR to verify the specificity and sensitivity of candidate biomarkers Sparc, Ifngr1 and Fapp5 in ischemic stroke with qi deficiency and blood stasis syndrome, and used ROC curves to quantify their diagnostic efficacy.

[0067] 1. Laboratory animals Forty-eight male SPF-grade SD rats (8 weeks old, weighing 230±20g) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed at the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. They were acclimatized for one week before the experiment.

[0068] 2 Experimental Groups After one week of acclimatization feeding, the rats were randomly divided into the following 6 groups: Control group: No action taken; MCAO group: The middle cerebral artery occlusion model was used to simulate the pathological state of simple ischemic stroke.

[0069] Qixu Xueyu Syndrome Group: Established a simple Qi deficiency and blood stasis syndrome model using a composite modeling method (exhaustion swimming + starvation).

[0070] Model group: The core model group of this study, based on the successful establishment of Qi deficiency and blood stasis syndrome, then performs MCAO surgery to simulate the state of clinical symptoms.

[0071] AD group: The Aβ1-42 hippocampal injection model was used as the disease control group to exclude the non-specific expression of the biomarker in other neurodegenerative diseases.

[0072] BYHW group: After the successful development of the ischemic stroke qi deficiency and blood stasis syndrome model, Buyang Huanwu Decoction was administered to observe the changes in the expression of candidate biomarkers after effective treatment and to verify their correlation with the syndrome type.

[0073] 3. Model Construction 3.1 MCAO Group The method is the same as in Example 1.

[0074] 3.2 Qixu Xueyu Syndrome Group The method is the same as in Example 1.

[0075] 3.3 Model Group The method is the same as in Example 1.

[0076] 3.4 AD group Bilateral hippocampal injection of Aβ 1-42 A rat AD model was established using the oligomer method. Rats were anesthetized via intraperitoneal injection, fixed on a stereotaxic instrument, and their skulls were exposed after shaving and disinfection. The coordinates of the CA1 region of the hippocampus were located posterior to the anterior fontanelle using a rat stereotaxic atlas. The skull was carefully opened with a micro-dental drill, and pre-aggregated Aβ was injected bilaterally at a slow rate (0.5 μL / min) using a microsyringe. 1-42 5 μL of oligomer solution, keep the syringe in place for 5 min.

[0077] 3.5 BYHW group After the model of ischemic stroke with qi deficiency and blood stasis was successfully established, the patient was administered the drug by gavage at a dose of 6.94 g / kg / 24h for 7 days.

[0078] 4. Sample Collection and Testing Rats were sacrificed at the experimental endpoint, and rat brain tissue was collected for ELISA and qPCR detection, using the same methods as in Examples 3 and 4. The diagnostic efficacy of the three candidate biomarkers for ischemic stroke with qi deficiency and blood stasis syndrome was objectively evaluated by plotting ROC curves and calculating AUC values, ultimately confirming their excellent differential diagnostic ability.

[0079] 5 Results 5.1 ELISA detection of Sparc, Ifngr1, and Fapp5 protein levels ELISA results showed that, compared with the Control group, MCAO group, Qixu Xueyu Syndrome group, Model group, AD group, and BYHW group, the protein expression levels of Sparc, Ifngr1, and Fabp5 in the brain tissue of the Model group were significantly upregulated. Figure 12 , P <0.01), while there was no significant change in expression among the MCAO group, Qixu Xueyu Syndrome group, and AD group. After intervention with Buyang Huanwu Decoction, the expression of all three proteins decreased compared with the Model group ( P <0.01), see Figure 12 .

[0080] 5.2 qPCR detection of Sparc, Ifngr1, and Fapp5 mRNA levels qPCR results showed that, compared with the Control group, MCAO group, Qixu Xueyu Syndrome group, AD group, and BYHW group, the transcription levels of Sparc, Ifngr1, and Fapp5 in the Model group were significantly increased. P <0.01), while there was no significant change in expression among the Model group, Qixu Xueyu Syndrome group, and AD group. Furthermore, after intervention with Buyang Huanwu Decoction, the abnormally high expression significantly decreased compared to the Model group ( P <0.01). This transcriptional trend is highly consistent with the protein level results observed by the aforementioned ELISA, jointly indicating that Buyang Huanwu Decoction can synergistically regulate the expression of Sparc, Ifngr1, and Fapp5 at both the gene transcription and protein translation levels. Figure 13 ).

[0081] 5.3 Assessment of the diagnostic efficacy of biomarkers To evaluate the potential of Sparc, Ifngr1, and Fapp5 as diagnostic biomarkers, we further plotted ROC curves ( Figure 14 The results showed that the area under the curve (AUC) for the Sparc diagnostic model was 0.80 (95% CI: 0.66–0.94), the AUC for the Ifngr1 diagnostic model was 0.89 (95% CI: 0.78–0.99), and the AUC for the Fapp5 diagnostic model was 0.84 (95% CI: 0.72–0.97). These results indicate that these three biomarkers have excellent differential diagnostic ability for ischemic stroke with qi deficiency and blood stasis syndrome. Figure 14). Example

[0082] Experiment 1: By specifically knocking down genes, we confirmed the key driving roles of Sparc, Ifngr1, and Fapp5 in the pathological process of Qi deficiency and blood stasis syndrome in ischemic stroke. Furthermore, by comparing the therapeutic effects of the classic formula Buyang Huanwu Decoction with those of specifically knocked-down genes, we explored the intrinsic connection between traditional Chinese medicine compound formulas and targeted therapy.

[0083] 1. Laboratory animals Forty-eight SPF-grade male SD rats, weighing 220±20g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed at the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. They underwent one week of acclimatization before the experiment.

[0084] 2. Experimental Grouping After one week of acclimatization feeding, the rats were randomly divided into 6 groups of 8 rats each.

[0085] Control group: Do nothing.

[0086] Model group: Based on the successful establishment of Qi deficiency and blood stasis syndrome, MCAO surgery was performed to simulate the state of Qi deficiency and blood stasis syndrome in ischemic stroke.

[0087] Model+BYHW group: After modeling ischemic stroke with qi deficiency and blood stasis syndrome, Buyang Huanwu Decoction was administered to observe its therapeutic effect.

[0088] Model + siSparc group: Based on the model of qi deficiency and blood stasis syndrome in ischemic stroke, Sparc was knocked down to observe the role of Sparc in qi deficiency and blood stasis syndrome in ischemic stroke.

[0089] Model + siIfngr1 group: Based on the ischemic stroke qi deficiency and blood stasis syndrome model, Ifngr1 was knocked down to observe the role of Ifngr1 in ischemic stroke qi deficiency and blood stasis syndrome.

[0090] Model + siFabp5 group: Based on the ischemic stroke qi deficiency and blood stasis syndrome model, Fapp5 was knocked down to observe the role of Fapp5 in ischemic stroke qi deficiency and blood stasis syndrome.

[0091] 3. Model Construction 3.1 Model Group The same method was used to construct the model of ischemic stroke with qi deficiency and blood stasis in Example 1.

[0092] 3.2 Model + BYHW Group After the model of ischemic stroke with qi deficiency and blood stasis was successfully established, the patient was administered the drug by gavage at a dose of 6.94 g / kg / 24h for 7 days.

[0093] 3.3 Model + siSparc group Sparc siRNA, 5-10 μg, dissolved in 10-20 μL buffer, is administered intrathecally.

[0094] 3.4 Model + siIfngr1 group Ifngr1 siRNA, 5-10 μg, dissolved in 10-20 μL buffer, is administered intrathecally.

[0095] 3.5 Model + siFabp5 group Use 5-10 μg of Fap5 siRNA dissolved in 10-20 μL buffer for intrathecal injection.

[0096] 4. Observation and detection indicators 4.1 Forelimb grip strength 4.2 Blood Rheology 4.3 Coagulation function 4.4 Neurological Function Scores 5 Results Compared with the control group, the forelimb grip strength of rats in the model group was significantly reduced. Figure 15 Neurological function scores significantly increased ( Figure 18 , P <0.01), the whole blood high-shear relative index, whole blood low-shear relative index, erythrocyte aggregation index and shear rate all increased significantly ( Figure 16 , P <0.05), while APTT and PT were significantly shortened ( Figure 17 , P <0.01 indicates that the syndrome of qi deficiency and blood stasis is clear.

[0097] All intervention groups reversed the above abnormalities to some extent. Compared with the Model group, the Model + BYHW group showed better recovery of grip strength. Figure 15 ), Improvement of neurological function ( Figure 18 ), hemorheology ( Figure 16 ) and coagulation function ( Figure 17 Significant improvements were observed in all aspects. P <0.05), confirming its overall therapeutic effect of invigorating qi and promoting blood circulation.

[0098] Notably, all three knockdown groups (Model + siSparc, Model + siIfngr1, Model + siFabp5) exhibited therapeutic effects, particularly in grip strength recovery. Figure 15 ), improve neurological function scores ( Figure 18 ), improve blood rheology ( Figure 16 ) and coagulation function indicators ( Figure 17 It has a significant effect in all aspects ( ) P <0.05).

[0099] In summary, Buyang Huanwu Decoction has a comprehensive effect on improving the qi deficiency and blood stasis syndrome in ischemic stroke. The three knockdown groups all exerted therapeutic effects at different pathological stages, suggesting that Sparc, Ifngr1, and Fapp5 play important roles in the qi deficiency and blood stasis syndrome in ischemic stroke.

[0100] Experiment 2: By constructing Sparc, Ifngr1, and Fapp5 overexpression models and directly comparing them with the Model group and the BYHW group, the different effects of targeted intervention and compound treatment were clarified, confirming that Sparc, Ifngr1, and Fapp5 are key pathogenic factors of Qi deficiency and blood stasis syndrome in ischemic stroke. Ultimately, these three factors were identified as biomarkers with both functional driving force and specificity in this syndrome.

[0101] 1. Laboratory animals Forty-eight SPF-grade male SD rats, weighing 220±20g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed at the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. They underwent one week of acclimatization before the experiment.

[0102] 2. Experimental Grouping After one week of acclimatization feeding, the rats were randomly divided into 6 groups of 8 rats each.

[0103] Control group: Do nothing.

[0104] Model group: Based on the successful establishment of Qi deficiency and blood stasis syndrome, MCAO surgery was performed to simulate the state of Qi deficiency and blood stasis syndrome in ischemic stroke.

[0105] Model + BYHW group: After modeling ischemic stroke with qi deficiency and blood stasis syndrome, Buyang Huanwu Decoction was administered to observe its therapeutic effect.

[0106] Model + AAV-Sparc group: Based on the ischemic stroke qi deficiency and blood stasis syndrome model, Sparc was overexpressed to observe the role of Sparc in ischemic stroke qi deficiency and blood stasis syndrome.

[0107] Model + AAV-Ifngr1 group: Based on the ischemic stroke qi deficiency and blood stasis syndrome model, Ifngr1 was overexpressed to observe the role of Ifngr1 in ischemic stroke qi deficiency and blood stasis syndrome.

[0108] Model + AAV-Fabp5 group: Based on the ischemic stroke qi deficiency and blood stasis syndrome model, Fap5 was overexpressed to observe the role of Fap5 in ischemic stroke qi deficiency and blood stasis syndrome.

[0109] Model + AAV-Sparc + BYHW group: In addition to the treatment given to the Model + AAV-Sparc group, BYHW decoction was administered.

[0110] Model +AAV-Ifngr1+BYHW group: In addition to the treatment given to the Model +AAV-Ifngr1 group, BYHW decoction was administered.

[0111] Model + AAV-Fabp5 + BYHW group: In addition to the treatment given to the Model + AAV-Fabp5 group, BYHW decoction was administered.

[0112] 3. Model Construction 3.1 Model Group The same method as in Example 1 for constructing the model of ischemic stroke with qi deficiency and blood stasis syndrome.

[0113] 3.2 Model + BYHW Group After the model of ischemic stroke with qi deficiency and blood stasis was successfully established, the patient was administered the drug by gavage at a dose of 6.94 g / kg / 24h for 7 days.

[0114] 3.3 Model + AAV-Sparc Group AAV-overexpression-Sparc virus was injected into the brain via stereotactic injection to specifically overexpress the Sparc protein.

[0115] 3.4 Model + AAV-Ifngr1 group AAV-overexpression-Ifngr1 virus was injected into the brain via stereotactic injection to specifically overexpress the Ifngr1 protein.

[0116] 3.5 Model + AAV-Fabp5 group AAV-overexpression-Fabp5 virus was injected into the brain stereotactically to specifically overexpress the Fap5 protein.

[0117] 3.6 Model + AAV-Sparc + BYHW Group Based on the Model + AAV-Sparc group, the drug was administered by gavage at a dose of 6.94 g / kg / 24h for 7 days.

[0118] 3.7 Model + AAV-Ifngr1 + BYHW group Based on the Model +AAV-Ifngr1 group, the drug was administered by gavage at a dose of 6.94 g / kg / 24h for 7 days.

[0119] 3.8 Model + AAV-Fabp5 + BYHW Group Based on the Model + AAV-Fabp5 group, the drug was administered by gavage at a dose of 6.94 g / kg / 24h for 7 days.

[0120] 4. Observation and detection indicators 4.1 Forelimb grip strength 4.2 Blood Rheology 4.3 Coagulation function 4.4 Neurological Function Scores 5 Results Compared with the control group, the forelimb grip strength of rats in the model group was significantly reduced. Figure 19 Neurological function scores significantly increased ( Figure 20 , P <0.01). In terms of hemorheology, the Model group showed significantly increased whole blood high shear rate, whole blood low shear rate, erythrocyte aggregation index, and shear rate. Figure 21 , P <0.05; Regarding coagulation function, APTT and PT were significantly shortened ( Figure 22 , P <0.01) indicates that the Model group has a hypercoagulable state and microcirculatory disturbance.

[0121] Compared with the Model group, the forelimb grip strength of rats in the Model +BYHW group was significantly restored. Figure 19 Neurological function scores significantly improved ( Figure 20 ), and at the same time, various blood rheological indicators ( Figure 21 ) and coagulation function (APTT and PT prolongation) Figure 22 All were effectively regulated. P The value of <0.05 once again confirms the efficacy of this formula in invigorating qi and promoting blood circulation.

[0122] It is noteworthy that in the three gene overexpression groups (Model + AAV-Sparc group, Model + AAV-Ifngr1 group, and Model + AAV-Fabp5 group), the forelimb grip strength in all three groups further decreased. Figure 19 ), with higher neurological deficit scores ( Figure 20 Abnormally elevated blood rheological parameters ( Figure 21 ) and further shortening of APTT and PT ( Figure 22 Both are more significant ( P <0.05). However, the combined administration of Buyang Huanwu Decoction on top of Model and overexpression failed to effectively reverse the above indicators. This result suggests that Sparc, Ifngr1, and Fapp3 may be key drivers of this disease, and their overactivation may mediate the pathological process to some extent and weaken the therapeutic effect of Buyang Huanwu Decoction.

[0123] 6. Identify biomarkers for Qi deficiency and blood stasis syndrome in ischemic stroke First, Buyang Huanwu Decoction has a clear therapeutic effect on rats with qi deficiency and blood stasis syndrome complicated by ischemic stroke. This formula can not only improve macroscopic symptoms of qi deficiency and blood stasis (such as weight loss, weakened grip, and purplish tongue), but also effectively reverse microscopic changes, including reducing the area of ​​cerebral infarction, promoting the recovery of nerve function, improving blood rheology and coagulation function, and repairing mitochondrial ultrastructural damage. The overall efficacy is dose-dependent.

[0124] Secondly, multi-omics integration analysis revealed the complex molecular basis of the disease and the targets of drugs. The study identified 406 differentially expressed proteins and 1032 differentially expressed genes, and further screened 25 common key targets with consistent expression at the protein and gene levels, providing important evidence for further exploration of biomarkers for qi deficiency and blood stasis syndrome in ischemic stroke.

[0125] Ultimately, this study identified Sparc, Ifngr1, and Fabp5 as potential biomarkers for the intervention of Buyang Huanwu Decoction in this disease. Experimental validation showed that the expression of all three was significantly upregulated at both the protein and transcriptional levels under model conditions, and Buyang Huanwu Decoction could reverse their abnormally high expression. Notably, the three knockdown groups also exhibited significant therapeutic effects in the relevant pathological processes, while the three overexpression groups did not show any therapeutic effect. Furthermore, even with the addition of Buyang Huanwu Decoction on the basis of overexpression, it failed to improve the symptoms of Qi deficiency and blood stasis syndrome, further supporting the key role of Sparc, Ifngr1, and Fabp5 in Qi deficiency and blood stasis syndrome in ischemic stroke from a functional perspective.

[0126] In summary, this study systematically elucidates, from overall animal phenotype and systems omics analysis to key target experimental verification, how Buyang Huanwu Decoction treats ischemic stroke with qi deficiency and blood stasis syndrome through multi-level regulatory networks. It also suggests the value of Sparc, Ifngr1, and Fapp5 as potential biomarkers, providing new scientific evidence for the research on the integration of disease and syndrome in traditional Chinese medicine.

Claims

1. A method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome, characterized in that, Includes the following steps: 1) Construct an animal model of ischemic stroke with qi deficiency and blood stasis syndrome; 2) Based on the model animals and control animals obtained in step 1), perform proteomics and transcriptomics analyses respectively to screen for differentially expressed proteins and genes; 3) Perform joint analysis on the differentially expressed proteins and genes obtained in step 2), and take the intersection molecules that differ at both the protein level and the transcription level as candidate biomarkers; 4) Verify the expression of the candidate biomarkers identified in step 3) in animal models and evaluate their diagnostic efficacy to identify biomarkers for ischemic stroke with qi deficiency and blood stasis syndrome.

2. The method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome as described in claim 1, characterized in that, The animal model in step 1) is the SD rat model.

3. The method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome as described in claim 1, characterized in that, Step 1) involves establishing a model of Qi deficiency and blood stasis syndrome through hunger and fatigue intervention, and then establishing an ischemic stroke model through middle cerebral artery occlusion.

4. The method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome as described in claim 1, characterized in that, In step 3), the joint analysis specifically involves taking the intersection of the gene corresponding to the differentially expressed protein and the differentially expressed gene to obtain common targets that show significant differences at both the protein and transcriptional levels.

5. The method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome as described in claim 1, characterized in that, In step 3), the identified candidate biomarkers include at least one of Mx1, Irf8, Cfd, Cd22, Pf4, Golm1, Pycard, Dnah12, Fgl2, Lcp2, Shc1, Sparc, Ifngr1, Tinagl1, Btk, Lrg1, Adh6, Rps19, Fapp5, Tlr3, Casp6, Fyb1, Nt5c, Fn1, and Usp4.

6. The method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome as described in claim 1, characterized in that, In step 4), the verification includes verifying the expression of the candidate biomarker at the protein level using enzyme-linked immunosorbent assay (ELISA), and / or verifying its expression at the mRNA level using quantitative polymerase chain reaction (PCR).

7. The method for screening and determining biomarkers in an animal model of ischemic stroke with qi deficiency and blood stasis syndrome as described in claim 1, characterized in that, In step 4), the assessment of diagnostic efficacy includes plotting the receiver operating characteristic curve and calculating the area under the curve.

8. The method for screening and determining biomarkers for an animal model of ischemic stroke with qi deficiency and blood stasis syndrome as described in claim 5, characterized in that, The candidate biomarkers are Sparc, Ifngr1, and Fapp5.

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