Effect of regulating qi and invigorate blood circulation method on hemorrheology in rats with acute blood stasis syndrome and its network pharmacology research

To observe the effect of regulating qi and invigorate blood circulation method on hemorrheology in rats with acute blood stasis syndrome from the perspective of modern pharmacology, and use network pharmacology to explain its mechanism. Forty male Wistar rats were randomly divided into 6 groups. Except the blank control group, the other groups were injected with adrenaline hydrochloride and ice bath to establish the acute blood stasis syndrome model. Each group were administered Xuefu Zhuyu D ecoction, Jinlingzi Powder, Taohong Siwu Decoction, and Yanhusuo Decoction, and t he blank control group and model control group were given the same volume of saline, respectively. The blood rheology indexes such as red blood cell aggregation index of rats were measured. The TCMSP and Batman database were used to search for chemical components and targets of Jinlingzi Powder, and protein interaction analysis, GO enrichment analysis and KEGG analysis were performed on the common targets. The experimental results show that, compared with the model group, the blood rheology index levels of the Yanhusuo group and Taohong Siwu Decoction group are reduced, while the blood rheology index levels of the Xuefu Zhuyu Decoction group and the Jinlingzi S an group are significantly reduced. The network pharmacology predicts that Jinlingzi Powder has 44 active ingredients and 34 drug-disease shared targets, and KEGG analyzes 61 signaling pathways. Conclusion: IN the case of blood stasis syndrome, the effect of regulating qi with invigorate blood circulation is better than that of regulating qi alone. Network pharmacologic prediction showed that TNF signaling pathway was strongly correlated with hemorheology, and its action pathway was closely related to key tar gets IL6, TNF, CASP3.


Introduction
Regulating Qi and invigorate Blood Circulation is one of the common treatments in the clinical practic e of traditional Chinese medicine. The combination of activating blood circulation medicine and regula ting qi medicine can regulate Qi mechanism, make Qi flow and promote blood circulation, and treat qi stagnation and blood stasis syndrome [1]. The theory stems from the theory of Qi and Blood in TCM. The medical doctors of the past generations used to treat blood stasis syndrome with this method. The There are many reports on the clinical research of the method of regulating qi and invigorate blood circulation, but few on its pharmacological experiment. Our research group based on the correlation be tween regulating qi and promoting blood circulation, this research group, taking Xuefu Zhuyu Decocti on and Jinlingzi powder as carriers, discusses the influence of Liqi Huoxue Method on Hemorheology of acute blood stasis model rats from the perspective of modern pharmacology, and clarifies its mecha nism by network pharmacology, and puts forward the scientificity and necessity of regulating qi and pr omoting blood circulation, so as to guide clinical application. Network pharmacology is based on the c haracteristics of traditional Chinese medicine in treating diseases with multiple targets and multiple lin ks. Its advantage is to break the defect of a single drug corresponding to a single disease. By searching relevant databases, combining data analysis, computer simulation and other methods to build a multi-le vel network, and finally realizes a method to predict the drug target as a whole and the mechanism of a ction [2][3].

Animal
Forty Wistar male rats were purchased from Chongqing Tengxin Biotechnology Co., Ltd., with a body weight of (220±10) g and a mouse age of (60±5) d. They were fed adaptively for one week. The temp erature of the laboratory is kept at about 25°C, and the rats are fed with basic feed, which is provided b y Chongqing Jinxin Co., Ltd. (which contains 16% protein, 8% fat, and 50% carbohydrate).

Instrument
SA-5600 automatic hemorheology tester, produced by Beijing Secide Technology Development Co., L td. (Model: SA-5600), Department of Laboratory, First Affiliated Hospital of Guiyang University of T raditional Chinese Medicine.

Animal grouping and administration method
Forty rats were randomly divided into blank control group, model control group, Taohong Siwu Decoc tion group (0.58 g/mL), Xuefu Zhuyu Decoction group (0.92 g/mL), Yanhusuo group (0.10 g/mL), Jinl ingzi powder group (0.20 g/mL). Rats in each group were administration the corresponding drug soluti on concentration, blank group and model group were given the same volume of normal saline with the volume of 2 mL/kg, each group was given once in the morning and evening.

Animal modeling and index detection
Rats were given the Chinese medicine decoction by intragastric administration. On the 6th day, all rats except the blank control group were injected subcutaneously with adrenaline hydrochloride (Adrenali ne, AD) injection 1.0 mg/kg, which was repeated once every 4 hours, and the interval was 2 hours. Aft er modeling, the rats in each group were fasted for 12 hours without water, and the blood was collected from the abdominal aorta with a single-use intravenous blood collection needle the next morning, and sent to the First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine for hemorh eology. Carcass viscosity, whole blood viscosity and plasma viscosity. Experimental data shows that th e blank control group and the model group have significant differences in the five indexes of red blood cell aggregation index, carcassone viscosity, whole blood high-cut, low-cut viscosity, and plasma visc osity P<<0.01, and each index of the model group is significantly increased, indicating successful mod eling. Compared with the blank control group, the model group * P<<0.01.

Statistical analysis
The experimental data was statistically analysed using SPSS17.0 software, and the data was expressed as mean ± standard deviation. One-way analysis of variance (One-Way ANOVA) was used to analyse the significance of the differences between the experimental data, and the multiple comparisons betwe en the means of the experimental data of each group (Multiple Comparisons). The results showed that P<<0.05 was significant difference and P<<0.01 was extremely significant difference.

Acquisition of active components in Jinlingzi powder
TCMSP [4] and Batman database were used to screen the chemical components of MeLia toosendanSi eb.et Zucc. And Corydalis yanhusuo W. T. Wang in Jinlingzi powder. The screening criteria of TCMS P database were oral bioavailability (OB) ≥ 30% and drug-like property (DL) ≥0.18. Searched the key words "Antithrombotic agents" and "hemorheology" in the Genecard database to download the targets related to hemorrheology, and integrate them to delete the duplicate targets. Use Veen's diagram (http:/ /jvenn.toulouse.inra.fr/app/example.html) to intersect the Jinlingzi scattered target and the hemorheolo gy target to obtain a common target. These common targets are there are 44 related to the chemical co mposition of Jinlingzi powder.

Establishment of a common target PPI network
By searching the String database, 34 common targets are imported, the species is set to "human", and a PPI network of key targets is constructed. Import the resulting TSV file into Cytoscape 3.6.1 software to obtain the topological parameter Degree, Betweenness centrality, and Closeness centrality values of each target. Filter the above three topological parameter values to meet the targets that are greater than all medians, and list them in the form of a table. Go out and visualize the top ten targets in the compreh ensive ranking.

Enrichment analysis of GO function and KEGG pathway
Use the DAVID (https://david.ncifcrf.gov/) database for the screened target targets for GO (Gene Onto logy) enrichment analysis and KEGG pathway analysis, and use KEGG Mapper (https://www.genome. jp /kegg/mapper.html) Display the target target in the channel.

Red blood cell aggregation index test results of each group
Compared with the erythrocyte aggregation index of the model group, the Xuefu Zhuyu Decoction Quanfang group and the Jinlingzi Powder group were significantly reduced (P<<0.01); the Taohong Siwu Decoction group and the Yanhusuo group were significantly reduced (P<<0.05), as shown in the table 2 shows. This indicated that Xuefu Zhuyu Decoction Quanfang and Jinlingzisan Quanfang were better than Taohong Siwu Decoction group and Yanhusuo group in improving the erythrocyte aggregation index of acute blood stasis rats.

Carson viscosity test results of each group
Compared with the carcass viscosity of the model group, the Xuefu Zhuyu Decoction Quanfang group and the Jinlingzi Powder group were significantly reduced (P<<0.01), and the Taohong Siwu Decoctio n group and the Yanhusuo group were significantly reduced (P<<0.05), as shown in Table 3. This indi cated that Xuefu Zhuyu Decoction Quanfang and Jinlingzisan Quanfang were better than Taohong Siw u Decoction group and Yanhusuo group in improving the carcass viscosity of acute blood stasis rats.

Test result of whole blood viscosity (200/s) of each group
Compared with the model group's whole blood high-cut viscosity (200/s), the Xuefu Zhuyu Decoction Quanfang group and the Jinlingzi Powder group were significantly reduced (P<<0.01); the Taohong Si wu Decoction group and the Yanhusuo group were significantly reduced (P<<0.05), as shown in Table  4. This indicated that Xuefu Zhuyu Decoction Quanfang and Jinlingzisan Quanfang were better than T aohong Siwu Decoction group and Yanhusuo group in improving the high blood viscosity (200/s) of a cute blood stasis rats.

Test result of whole blood viscosity (1/s) of each group
Compared with the model group's whole blood low-cut viscosity (1/s), the Xuefu Zhuyu Decoction Qu anfang group and the Jinlingzi Powder group were significantly reduced (P<<0.01), and the Taohong S iwu Decoction group and the Yanhusuo group were significantly reduced (P <<0.05), as shown in Tab le 5. This indicated that Xuefu Zhuyu Decoction Quanfang and Jinlingzisan Quanfang were better than Taohong Siwu Decoction group and Yanhusuo group in improving the low blood viscosity (1/s) of ac ute blood stasis rats.

Test results of plasma viscosity of each group
Compared with the plasma viscosity of the model group, the Xuefu Zhuyu Decoction Quanfang group and the Jinlingzi Powder group were significantly reduced (P<<0.01), and the Taohong Siwu Decoctio n group and the Yanhusuo group were significantly reduced (P<<0.05), as shown in Table 6. This indi cated that Xuefu Zhuyu Decoction Quanfang and Jinlingzisan Quanfang were better than Taohong Siw u Decoction and Yanhusuo in improving the plasma viscosity of acute blood stasis rats.  From the statistics of the above data, it can be seen that for the improvement of various indexes of b lood rheology in rats with acute blood stasis, the effect of combining blood circulation drugs with liqi drugs is the best. The experimental results show that the Jinlingzi San group and the Xuefu Zhuyu Dec oction group have a good effect on improving the blood rheology indexes of acute blood stasis model r ats (P<<0.01). Tang network pharmacology reports, and the research group mainly focuses on Jin Ling zi powder. Therefore, in order to conduct a deeper research on the follow-up Jinlingzi Powder, the auth or conducted a network pharmacological prediction of Jinlingzi Powder, with a view to providing direc tion for the next experiment.

Active target and disease screening
In the Genecards database, using "Antithrombotic agents" and "hemorheology" as keywords, a total of 456 targets related to hemorheology were retrieved, and 292 targets related to Jinlingzi. The screening results changed the blood rheology Match the learning target with the related targets of Jinlingzisan and draw a Venn diagram, as shown in Figure 1. There are 34 common targets, indicating that these 34 targets participate in the common system of the active ingredient of Jinlingzi Powder-hemorheology.

Construction of key target PPI network
Search the String database online, import 34 intersection targets, set the species "human", and take the confidence = 0.400, where number of nodes = 34, number of edges = 151, average node degree = 8.88, avg. local clustering coefficient = 0.745, Expected number of edges = 41, PPI enrichment p-value <<1. 0e-16, the results are shown in Figure 2. The results are imported into Cytoscape 3.6.1 software in TSV format, and the three topological parameters are calculated by obtaining the topological parameters De gree (node connection degree), Betweenness centrality (node connection degree), and Closeness centra lity (node tightness degree) of each target. The medians are 9, 0.00877577 and 0.56603774, respectivel y. Screening targets with Degree, Betweenness centrality, and Closeness centrality greater than the me dian, a total of 12 key targets were obtained, and 12 targets were displayed as data, as shown in Table  7. The relationship between the top ten targets in the comprehensive ranking is shown in the network d iagram. As shown in Figure 2, the darker the red, the higher the ranking and the stronger the correlatio n.

Enrichment analysis of GO biological functions
A total of 133 biological processes, 14 cellular processes, 26 molecular processes, and the first 5 biological processes were analyzed by GO enrichment analysis on 34 common targets of Jinlingzi power composite target and hemorheology-related targets Enrichment includes inflammatory response, response to lipopolysaccharide, response to hypoxia, cellular response to lipopolysaccharide, response to amino acid. Quick Go Inflammatory response means the immediate defense of vertebrate tissues against infection or injury caused by chemical or physical substances, while response to lipopolysaccharide is any process of state or activity (movement, secretion, enzyme production, gene expression, etc.) induced by LPS stimulus. By screening P<<0.005, 35 biological processes were obtained, and the first 20 biological processes were presented in Table 5. It reflects that the mechanism of action between Jinlingzi powder and hemorheology involves many biological processes in the body, and each biological process is interrelated.

KEGG pathway analysis
34 potential targets were mapped into the database for KEGG pathway enrichment analysis, a total of 6 1 signal pathways were obtained. KEGG enrichment analysis P≤0.005, screened out 27 signal pathway s mainly enriched by key targets. These pathways are closely related to the mechanism of action of Jinl ingzi Powder and hemorheology, including tumor necrosis factor signaling pathway, malaria, African t rypanosomiasis, trypanosomiasis (American trypanosomiasis), amoebiasis, and Influenza and other pat hways. The first 20 channels with significant differences are output in the form of a bubble chart, the r esults are shown in Figure 5. Cytoscape 3.6.1 software was used to visually analyze 34 common targets, diseases, active ingredie nts and drugs, and an interactive network of Jinlingzi powder and hemorheology was constructed [5][6]. After screening out the corresponding interacting proteins and visualizing them with different colors a nd shapes, you can intuitively see the network relationship between the active chemical components an d the target. There are 34 round green genes, representing the disease-drug shared genes, 44 in the inve rted triangle, representing the active ingredients of Jinlingzi powder, red is the disease, the prism is the medicine composed of Jinlingzi powder, and purple is the golden bellflower The results show that the greater the degree of connectivity, the greater the shape, as shown in Figure 6.

Discussion
According to Traditional Chinese medicine, blood stasis refers to stagnant or stagnant blood flow, form ing "evil blood", "sepsemia" and "blood leaving the meridian", which is blocked in the meristem and z ang-fu organs and causes a series of diseases, namely blood stasis syndrome, or blood stasis syndrome [7], which involves a wide range of diseases, including internal and external diseases, women, childre n, injuries and other diseases [8].
Aiming at the blood stasis syndrome, the principle of "relieving its qi and blood, allowing it to be a djusted, and bringing peace" [9] is emphasized during treatment, that is, treating blood and regulating q i. The relationship between qi and blood in Traditional Chinese medicine is often described as "Qi is th e leader of blood" and "blood is the root of qi". "Qi is the leader of blood" includes qi can produce blo od, Qi can make blood and Qi can drink blood. Among them, qi can make blood closely related to bloo d stasis, that is, the circulation of blood in the veins depends on the promotion of Qi, that is, "Qi can m ake blood, qi stagnation means blood stasis". "Theory of Blood Syndrome Vomiting Blood" [10] Clou d: "Qi knot leads to blood coagulation"; "Where there is blood stasis, not only blocked the airway, bloc k vitality". The promotion of heart qi, the publicity and dissemination of lung qi, and the dredge of live r qi are all closely related to the operation of blood. No matter which link is dysfunctional, it can lead t o the obstruction of blood flow. If blood stasis blocks the arteries and veins, the blood running is not s mooth, it will also affect the normal drainage of qi, which is manifested as qi stagnation. The two influ ence each other, and see the evidence of qi stagnation and blood stasis. Therefore, the treatment should promote blood circulation and regulate qi.
Objective indicators in the diagnosis of blood stasis syndrome are all related to blood circulation, th at is, vessels, blood and blood flow and their interactions. In a broad sense, they are all closely related t o hemorheology, which has become one of the important and practical means for the diagnosis of bloo d stasis syndrome and the study of promoting blood circulation and removing blood stasis [11]. Wu Xi aomei et al [12] study of blood stasis syndrome is associated with the changes in the hemodynamics an d microcirculation, blood flow in vascular, cycling, as a fluid, when its rheological properties change, mainly in predominantly blood viscosity abnormal hemorheology indexes, all kinds of promoting bloo d circulation to remove blood stasis of TCM compound also had an improved the effect of blood rheol 12 ogical property. Whole blood high-cut, low-cut viscosity, all high, indicating that the whole blood visc osity increased. Whole blood viscosity refers to the viscosity of a liquid. The greater the viscosity, the slower the flow. Conversely, the faster the flow. Increased whole blood viscosity indicates increased h ematocrit or plasma viscosity, increased aggregation of red blood cells, poor deformability or elasticity of red blood cells, and hardened and rough blood vessel walls, causing increased resistance to blood fl ow, slowing blood flow velocity, and finally leading to blood flow stagnation and formation "Blood sta sis", a disorder of blood circulation and microcirculation in the whole body or part [13], directly affect s the blood supply to the organs, resulting in disease. Experiments have shown that Jinlingzi Powder h as a good therapeutic effect on hemorrheology, but the mechanism of Jinlingzi Powder's intervention i n hemorrheology is still unclear. According to online pharmacological predictions, Jinlingzi powder is mainly composed of Jinlingzi (L. chinensis) and Yanhusuo. The chemical components of Jinlingzi are Mandenol, Ethyl linolenate, etc., and the chemical components of Yanhusuo are Isocorypalmine, bicuc ulline, quercetin. Among them, studies have reported that quercetin combined with adenosine diphosp hate and thromboxane A inhibitor can effectively inhibit platelet extension [14]. These active compone nts related to hemorheology are mainly related to IL6, AKT1 CASP3 and other genes on the TNF sign aling pathway. There are also reports of TNF signaling pathway related to hemorheology and other dis eases [15][16], as shown in Figure 7. Pharmacodynamics experiments and network pharmacology prediction results show that after mode ling, the five indexes of erythrocyte aggregation index, carcass viscosity, whole blood high-cut, low-cu t viscosity, and plasma viscosity are significantly increased, indicating that the model of blood stasis m odel success. After medication, the blood indexes of rats in each group were significantly reduced exce pt for the blank control group and the model group. use of blood circulation drugs and qi-regulating drugs can reduce the index levels of whole blood visco sity in hemorheology, and the two types of drugs are effective when used alone. The combination of H uoxue medicine and Liqi medicine can reduce the level of whole blood viscosity and other indicators, t hat is, Xuefu Zhuyu Decoction and Jinlingzi San have a significant improvement in blood flow in acut e blood stasis rats. The effect of metamorphic index is better than that of blood circulation medicine or Qi medicine alone. Network pharmacological predictions indicate that TNF signaling pathway is closel y related to hemorheology. Tumor necrosis factor (TNF), as an important cytokine, can induce multipl e signaling pathways in the cell, including apoptosis, Cell survival, inflammation and immunity. After being activated, TNF is assembled into homotrimers, resulting in trimerization of TNFR1 or TNFR2 [1 7]. Almost all cells express TNFR1, which is the main receptor of TNF (also known as TNF-alpha). In contrast, TNFR2 is expressed in limited cells, such as CD4 and CD8 T lymphocytes, endothelial cells, microglia, oligodendrocytes, neuronal subtypes, cardiomyocytes, thymocytes, and human mesenchym e stem cell. It is a receptor for TNF and LTA (also known as TNF-beta). When binding to a ligand, TN FR mediates the binding of some adapter proteins (such as TRADD or TRAF2), which in turn initiates the recruitment of signal transducers [18]. It can be deduced from this: ① the use of blood-activating drugs or qi-regulating drugs alone can reduce the level of blood rheology indicators of the blood stasis model, and can help the blood stasis dissipate and improve the blood flow. In particular, the applicatio n effect of the medicine for regulating qi proves that the theory of "qi leads to blood" in Traditional Ch inese medicine is correct and reasonable. ②The effect of reducing the index level is more clear and sig nificant after the compatibility of activating blood circulation and regulating qi, indicating that the use of activating blood circulation is more effective than the use of activating blood circulation or regulatin g Qi alone. Therefore, in various types of blood stasis syndrome, especially in the high incidence of ca rdiovascular diseases, the results of the experimental research of our research should be able to better g uide clinicians to use the combination of qi and activating blood therapy to treat clinical blood stasis T he card provides an important treatment method and also lays the foundation for the follow-up research of Jinlingzi powder.