Qi-tonifying and blood-activating formula for improving diabetic vascular complications and application thereof

Abstract

The invention relates to a qi-tonifying and blood-activating formula for improving diabetic vascular complications and application thereof.The qi-tonifying and blood-activating formula is prepared from astragalus membranaceus, radix rehmanniae recen, the root of kudzu vine, ligusticum wallichii and radix trichosanthis, and the prepared qi-tonifying and blood-activating formula is verified and analyzed through pharmacological network construction of a model sample; the obtained qi-tonifying and blood-activating formula can improve the blood sugar of diabetic rats, reduce the levels of ICAM-1 and VCAM-1, improve vascular tissue damage and improve the cell viability and migration ability of HUVEC in a high-glucose environment, and the action mechanism of the formula is exerted by adjusting an MAPK signal channel, so that diabetic vascular complications can be improved.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine, and in particular to the field of a Qi-invigorating and blood-activating prescription for improving diabetic vascular complications. Background Art

[0002] Diabetes mellitus (DM) is a chronic progressive endocrine metabolic disease. With the continuous development of the world economy and the improvement of people's living standards, the prevalence of diabetes in my country has also shown an increasing trend year by year. As the course of diabetes increases year by year, it will cause a series of complications such as liver, kidney, and heart. Among the many complications, one of the main causes of death in diabetic patients is macrovascular disease, which accounts for about 70%-80% of diabetes-related deaths. Therefore, preventing and delaying vascular complications has become an urgent problem to be solved. The mechanism leading to diabetic vascular complications is very complex, among which endothelial damage is considered to be an early indicator of diabetic macrovascular complications. Vascular endothelial cells are one of the main components of the intravascular environment. When vascular endothelial cells are impaired, a variety of cardiovascular diseases will occur. Recent studies have shown that high concentrations of glucose can promote the overexpression of cell adhesion molecules (CAMs), including intracellular adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) on the surface of endothelial cells, and bind to circulating immune cells, leading to leukocyte recruitment, thereby aggravating endothelial dysfunction.

[0003] Modern medicine has found that glucose and lipid metabolism disorders and the resulting vascular endothelial damage are the main factors and pathological basis of diabetic vascular complications. Risk factors such as elevated blood sugar and dyslipidemia can cause macrophages and vascular smooth muscle cells to phagocytize a large amount of lipids to form foam cells, aggravating the inflammatory response of the blood vessels and leading to vascular endothelial damage and endothelial dysfunction. The United Kingdom Prospective Study (UKPDS) and the United States Diabetes Complications and Control Trial (DCCP) also confirmed that hyperglycemia is the main factor in chronic complications of diabetes. Fang Zhaohui et al.

[17] used the method of invigorating qi, activating blood circulation and nourishing yin to treat 35 DCHD patients. The results showed that this method can not only significantly reduce the clinical signs of patients, but also significantly improve the glucose and lipid metabolism of diabetic patients and prevent the occurrence and development of complications. Liu Cailing used the method of invigorating qi, activating blood circulation and nourishing yin to treat 26 cases of diabetes. The results showed that fasting blood sugar and glycosylated hemoglobin (HbAlc) in the treatment group were significantly reduced. Wu Jiping et al. used Danzhi Jiangtang Capsule, which has the effects of invigorating qi, activating blood circulation and nourishing yin, to intervene in patients with diabetes and macrovascular disease. The results showed that compared with the control group, Danzhi Jiangtang Capsule could effectively improve the postprandial blood glucose, HbA1c and endothelin levels of the patients in the treatment group, indicating that Chinese medicine capsules with the effects of invigorating qi, activating blood circulation and nourishing yin can improve glucose metabolism and repair vascular endothelial function. A large number of studies have shown that Shenqi compound prescription and its modified prescriptions, which have the effects of invigorating qi, activating blood circulation and nourishing yin, can regulate blood lipid biochemical indicators, reduce the damage of high sugar and high fat to blood vessels, and thus play a protective role in blood vessels.

[0004] Traditional Chinese medicine has unique insights into the diagnosis and treatment of diseases. According to the symptoms of diabetes, "three mores and one less", traditional Chinese medicine classifies it as "xiaoke". Diabetic vascular complications are a modern disease name, and according to its clinical manifestations, they can also be classified as xiaoke. Its etiology and pathogenesis are due to deficiency of both qi and yin, and the excess of dryness and heat, which leads to blood stasis blocking the collaterals. The treatment should be to replenish qi and nourish yin, and to activate blood circulation and remove blood stasis. A large number of studies have shown that the method of replenishing qi and activating blood circulation can effectively prevent and treat diabetic vascular lesions, and delay the progression of diabetic vascular complications from aspects such as improving sugar metabolism, reducing oxidative stress and inflammatory response. Summary of the invention

[0005] The technical purpose of the present invention is to solve the problems in the background technology and to provide a Qi-invigorating and blood-activating prescription for improving diabetic vascular complications and its application.

[0006] The technical purpose of the present invention is achieved through the following technical solutions: The Yiqi Huoxue recipe is made up of Astragalus, Radix Rehmanniae, Radix Puerariae, Rhizoma Chuanxiong and Radix Trichosanthis. The Yiqi Huoxue recipe is tested in vitro and in vivo, and the pharmacological network of the model sample is constructed to verify the characterization. Preparation process of Yiqi Huoxue prescription: weigh appropriate amount of astragalus, raw rehmannia, kudzu root, chuanxiong and trichosanthes root into a flask, add water to cover 3cm and soak for 1h, decoct twice after soaking, filter and rotary evaporate to concentrate to 0.85-1.20g / mL and freeze for later use; the amount of medicinal materials is 20-30g of astragalus, 5-15g of raw rehmannia and kudzu root, 3-8g of chuanxiong and trichosanthes root.

[0007] The effect verification steps of the Yiqi Huoxue recipe are as follows: (1) Preparation of other related reagents: Metformin solution, 3% pentobarbital, streptozotocin (STZ) solution required for in vivo experiments, and 3% pentobarbital solution, electrophoresis buffer, transfer buffer, TBST solution, blocking buffer, cell lysis buffer, BCA protein quantitative colorimetric solution, and DMEM culture medium required for in vitro experiments. (2) In vitro and in vivo experiments: In vivo experiment: SPF male SD rats were selected, 5-10 rats were randomly selected as the normal group, and the rest of the rats were injected with streptozotocin STZ once by tail vein injection to induce T1DM rat model; after the model was successfully established, the rats were randomly divided into model group, positive drug group (metformin group), and Qi-invigorating and blood-activating group (Qi-invigorating and blood-activating prescription group), with 8 rats in each group, and the drug was administered for 4 weeks; the rats were weighed once a week and fasting blood glucose FBG was measured once; after 4 weeks of administration, the rats were anesthetized, serum was collected, and the levels of ICAM-1 and VCAM-1 in each group of rats were detected by ELISA; the aortic arch of the rats was collected and stored in 4% paraformaldehyde, and HE staining was performed to observe its histopathological changes; In vitro experiments: After human umbilical vein endothelial cells (HUVECs) were resuscitated, they were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), and models were established using DMEM medium with a glucose concentration of 50 mmol / L. After successful model establishment, the MTT method was used to detect the effects of different concentrations of drug-containing serum on cell viability to select the optimal drug-containing serum concentration. A cell scratch experiment was performed to detect the effects of drug-containing serum on high-glucose-induced HUVECs. Network pharmacology was used to analyze the targets of the Yiqi Huoxue recipe on diabetic vascular damage, and the Western blot (WB) method was used in vitro to verify its mechanism. (3) Data processing and result analysis: Analyze the test results of (2).

[0008] It is composed of raw astragalus, Chuanxiong, Pueraria root, raw rehmannia and Radix Trichosanthis. In the formula, astragalus nourishes the spleen and replenishes qi, produces body fluid and quenches thirst, makes the spleen ascend and disperse, disperses essence to the lungs, and transports and distributes body fluids, which is the main medicine; raw rehmannia clears heat and cools blood, nourishes yin and produces body fluid, nourishes kidney yin to reduce virtual fire, and nourishes yin body fluid to clear latent heat, which is the minister medicine. Chuanxiong promotes qi and blood circulation, disperses blood stasis, and is a key medicine for qi stagnation and blood stasis, which is the minister medicine. Pueraria root produces body fluid and quenches thirst, and leads raw rehmannia into the Yangming meridian to treat the internal heat in the middle jiao of Yangming. At the same time, it helps astragalus to promote the spleen and stomach to clear yang, transport and disperse body fluids and quench thirst, which is the minister medicine and the meridian-leading medicine. Trichosanthes is used to nourish yin and clear heat, moisten dryness and quench thirst. On the basis of replenishing qi and nourishing yin, the whole formula adds the method of promoting blood circulation and unblocking collaterals, which complement each other and achieve the treatment of both the symptoms and the root causes.

[0009] Preferably, the solutions in (1) are prepared as follows: Preparation of metformin: Weigh sufficient amount of metformin sustained-release tablets as needed, add 0.5% sodium carboxymethyl cellulose solution to make the final concentration 3 mg / mL, and obtain metformin solution after full dissolution by ultrasonication; Preparation of streptozotocin: Weigh a sufficient amount of STZ powder under light-proof conditions, and dissolve STZ in 0.1 mol / L citric acid buffer with a pH of 4.5 to form a 2% STZ solution; Preparation of 3% pentobarbital: Weigh the required amount of pentobarbital solid, dissolve it in 0.9% sodium chloride solution to obtain a 3% pentobarbital solution and store it at 4°C away from light; Preparation of electrophoresis buffer: accurately weigh Tris base, SDS and Glycine in a ratio of 3:1:14.4, put them into a beaker and add appropriate amount of pure water, shake the liquid to fully dissolve it, continue to add pure water to make up to 1000 mL, and store in a 4℃ refrigerator for later use; Preparation of transfer solution: accurately weigh 3.00 g Tris base and 14.50 g Glycine, place in a beaker, add a little pure water, shake and mix, add 200 mL ethanol, and then make up to 1000 mL. Transfer the prepared transfer solution to a glass bottle and store in a 4°C refrigerator for later use; Preparation of TBST solution: weigh 2.42 g tris base and 8.00 g NaCl into a beaker, add a little pure water, shake the liquid to mix it thoroughly, continue to add pure water to make the volume 1000 mL, and adjust the pH value to above 7.5 with concentrated hydrochloric acid; after the solution is transferred to a glass bottle, add 1 mL of Tween-20, wait for it to be fully mixed and store it in a refrigerator at 4°C for later use; Blocking solution: 5% TBST skimmed milk powder solution; Preparation of cell lysate: Take out Enhanced RIPA Lysis Buffer in advance and place it on ice to fully dissolve it. Add 30.4 μL PMSF to every 2 mL Enhanced RIPA Lysis Buffer to prepare an appropriate amount of lysate to a final concentration of 1 mM. Preparation of BCA protein quantitative colorimetric solution: According to experimental needs, mix BCA reagent A and BCA reagent B in a ratio of 50:1 to prepare an appropriate amount of BCA working solution; Preparation of 50 mmol / L DMEM medium: Add anhydrous glucose powder to the cell solution and make up to 1 mL to prepare a glucose solution with a concentration of 3 mmol / L. Filter the glucose solution using a microporous filter membrane (0.22 μm), and then add the glucose solution to 50 mL of high-glucose DMEM medium to obtain 50 mmol / L DMEM medium, which is stored in a 4°C refrigerator.

[0010] As a preferred method, (2) in vivo experimental diabetic rat model establishment: select several SD rats with a body weight range of 260-280g and an age of 6-8 weeks, and have free access to food and water during the feeding period; after 2 weeks of adaptive feeding, the rats are fasted but not watered for 14 hours, and their fasting blood glucose and body weight are tested and recorded at this time; streptozotocin (STZ) is used to establish the model, and a single tail vein injection of STZ at 55 mg / kg is given according to the rat's body weight, and the rats continue to fast for 2 hours after the injection; after the injection of STZ, fasting blood glucose is tested on the 3rd, 7th and 10th days, and rats with blood glucose greater than 16.7 mmol / L are selected as stable models to proceed to the next step of the experiment; (2) The process of establishing the high-glucose-induced HUVEC cell injury model described in the in vitro experiment is as follows: HUVECs in the culture dish are revived, cryopreserved after subculturing, and the cells are resuspended in high-glucose DMEM medium containing 10% FBS and diluted to a density of 3×10 4 / mL of cell suspension; add 100 μL of cell suspension to each well of a 96-well plate, tap the plate to evenly distribute the cells; place the plate in a cell culture incubator and culture overnight to allow the cells to adhere to the wall; after ensuring adhesion, discard the culture medium, add culture medium with different glucose concentrations, and place the plate in an incubator for culture for 24-48 h.

[0011] As a preferred method, (2) in vivo experimental index detection and HE staining thoracic aorta tissue morphology observation; wherein the index detection includes general physical sign observation, body weight recording, blood sugar detection and biochemical index detection; the biochemical index detection method is that the rats in each group are killed at the end of the 4th week of drug administration, and relevant specimens are collected. The rats are fasted but not watered for 12 hours before being killed, and 3% pentobarbital is used for intraperitoneal injection to anesthetize the rats, and then blood is collected from the abdominal aorta. The collected blood is allowed to stand for 30 minutes and then centrifuged at 3000-5000rpm for 15 minutes. The supernatant is aspirated to obtain serum, and the ELISA kit is used to detect the content of VCAM-1 and ICAM-1 in rat serum, and the corresponding biochemical index levels are calculated.

[0012] Preferably, the HE staining thoracic aorta tissue morphology observation process is to remove the rat heart and aortic arch part, peel off the surrounding connective tissue, fix it in 4% paraformaldehyde for 24 hours, dehydrate and make it transparent, and then slice it according to the sequence of hematoxylin for 5 minutes, 0.1% hydrochloric acid alcohol for 10 seconds, pure water washing 2-5 times, 0.6% ammonia water blueing for 2-5 seconds, running water washing, and eosin staining solution for 3 minutes. The stained slices are then dehydrated, sealed and observed under an optical microscope.

[0013] Preferably, the in vitro MTT method in (2) is as follows: after the culture is completed, 50 μL of MTT reagent (2 mg / mL) is added to each well under light-proof conditions, and the well is placed in an incubator for incubation for 4 h; after the incubation is completed, the liquid in the well is discarded and the well is gently tapped on a clean toilet paper to completely absorb the liquid in the well; 150 μL of DMSO reagent is added to each well and the well is placed on an oscillator for 5-10 min; after the cell crystals are completely dissolved, the well is placed in an ELISA reader, and the absorbance value is measured at a wavelength of 490 nm to calculate the relative survival rate of each group of cells. The formula is: relative cell survival rate = (OD490 high glucose group - OD490 blank group) / (OD490 normal group - OD490 blank group).

[0014] As a preferred method, the cell scratch test process in (2) is as follows: resuspend the cells in high-glucose DMEM medium containing 10% FBS and dilute to a density of 3×10 4 / mL cell suspension; add 2mL of cell suspension to each well of a 96-well plate, tap the well plate to evenly distribute the cells; place the well plate in a cell culture incubator and culture overnight to allow the cells to adhere to the wall; after confirming the adhesion, discard the culture medium, add PBS to wash the residual culture medium, place it in an incubator and culture it for 24-48 hours, then add high-glucose DMEM culture medium containing 2.5% blank rat serum and 7.5% drug-containing serum and place it in an incubator for intervention for 48 hours, after which the liquid in the well is discarded and the well is scratched with a pipette tip, PBS is added, and then the excess cells are washed off; the cell scratches obtained are observed under an inverted microscope, and the cell migration is observed and photographed under an inverted microscope at 0 h, 6 h, 12 h, and 24 h after scratching, and the area difference between 0 h and 24 h is determined by Image J.

[0015] Preferably, the network pharmacology analysis process is as follows: after screening the ingredients of the Yiqi Huoxue prescription, drug target prediction and disease target collection are performed, after obtaining the common targets of drugs and diseases, protein interaction PPI network construction and enrichment analysis are performed, and finally, a "traditional Chinese medicine-ingredient-target-pathway-disease" network is constructed based on the relationship between protein targets and KEGG signaling pathways.

[0016] Preferably, the Western blot method is used to detect protein levels by extracting total protein from cells at the end of the intervention, determining protein concentration using the BCA method and preparing protein samples, loading protein samples for electrophoresis and incubating with antibodies, and finally performing imaging and subsequent data analysis. All statistical analyses were performed using SPSS 22.0 software and GraphPad Prism 8.4.3 software.

[0017] The advantage of adopting the present invention is that, combined with the verification method of the effect of the above-mentioned Yiqi Huoxue prescription on hyperglycemia, it can more deeply and intuitively demonstrate the great significance of the Yiqi Huoxue prescription of the present invention in regulating the MAPK signaling pathway to improve diabetic vascular complications.

[0018] In summary, the present invention has the following beneficial effects: 1. The method of improving diabetic vascular complications by regulating the MAPK signaling pathway with a Qi-invigorating and blood-activating prescription of the present invention, using the theory of traditional Chinese medicine, according to the etiology and pathogenesis of diabetes mellitus with yin deficiency, hyperactivity of fire, and endogenous dryness and heat, combines the classic prescription Siwu Decoction with Yuye Decoction to formulate a Qi-invigorating and blood-activating prescription. In the prescription, Astragalus is the main drug, Rehmannia root and Chuanxiong are the ministerial drugs, Radix Trichosanthis is the adjuvant drug, and Pueraria root is the guiding drug. Each medicinal material complements each other, and on the basis of invigorating Qi and nourishing Yin, the effect of promoting blood circulation and removing blood stasis is added to achieve the purpose of treating both the symptoms and the root causes; 2. Construct diabetic animal models and high-glucose damaged cell models, use in vivo and in vitro experiments to explore the effects of diabetes on vascular diseases, use the advantages of network pharmacology to build a holistic network relationship diagram, and demonstrate the therapeutic targets, biological processes and related mechanisms of the Yiqi Huoxue recipe on diabetic vascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the body weight changes of rats in each group over 4 weeks; Figure 2 shows the FBG levels of rats in each group at 4 weeks; Figure 3 shows the levels of VCAM-1 and ICAM-1 in rats of each group; Figure 4 is a representative picture of aortic arch pathological sections of rats in each group; Figure 5 shows the results of high glucose intervention on HUVEC cells for 24h and 48h; Figure 6 shows the effects of different concentrations of drug-containing serum on HUVECs intervened by high glucose; FIG7 shows the effect of drug-containing serum on HUVEC migration ability; Figure 8 is a Venn diagram of the relationship between drug and disease targets; FIG9 is a protein-protein interaction network diagram of the target of Yiqi Huoxue prescription in treating diabetic vascular disease; Figure 10 is a secondary classification bar chart of GO enrichment analysis (Top 10); Figure 11 is a bubble chart of KEGG enrichment analysis (Top 10); Figure 12 is a network diagram of “traditional Chinese medicine-ingredient-target-pathway-disease”; FIG13 shows representative images and quantitative analysis of JNK, ERK, p38 and p-JNK, p-ERK, and p-p38 in each group of cells; Among them, Figure 7 (A) is a representative picture of the cell migration experiment; (B) is the cell migration rate; Figure 13 (A) is a representative picture of different proteins in each group of cells; (B) is the quantitative analysis of different proteins in each group of cells. DETAILED DESCRIPTION

[0020] The following specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

[0021] It should be noted that the formula of the Qi-invigorating and blood-activating prescription adopted in this embodiment is 24g of Astragalus, 12g each of Rehmannia root and Pueraria root, 6g each of Chuanxiong and Radix Trichosanthis, and other related reagents are prepared uniformly, and its preparation method and other axioms and common sense are not elaborated in detail.

[0022] In vivo experiments: Prepare the Yiqi Huoxue recipe and other related reagents, and select 40 SD rats with a body weight range of 260-280g and an age of 6-8 weeks. During the feeding period, the rats were free to eat and drink water. After 2 weeks of adaptive feeding, the rats were fasted for 14 hours but not water, and the fasting blood glucose and body weight at this time were detected and recorded. Streptozotocin (STZ) was used to establish the model, and STZ was injected into the tail vein once at 55 mg / kg according to the body weight of the rats, and the rats continued to fast for 2 hours after the injection. After the injection of STZ, fasting blood glucose was tested on the 3rd, 7th, and 10th days, and rats with blood glucose greater than 16.7 mmol / L were selected as stable models. Eight normal SD rats were randomly selected as the normal group according to body weight, and 24 rats were selected from the rats with successful modeling and randomly divided into 3 groups according to body weight and blood glucose, namely the model group, Yiqi Huoxue group (Yiqi Huoxue recipe group), and the positive drug group (metformin group). The Qi-Yi-Huoxue group was given 10.8 g / kg of the Qi-Yi-Huoxue prescription every day according to body weight, the positive drug group was given 30 mg / kg of metformin every day according to body weight, and the normal group and the model group were given an equal amount of pure water at the same time, for four consecutive weeks.

[0023] The coat color, mental state, activity, daily diet and water intake, and excretion status of the rats were observed. All rats were weighed once a week and their body weights were recorded. After successful modeling and drug administration, the 12-hour fasting blood glucose of the rats was tested once a week.

[0024] Rats in each group were killed at the end of the 4th week of drug administration, and relevant specimens were collected. Rats were fasted but not watered for 12 hours before being killed. 3% pentobarbital was injected intraperitoneally to anesthetize the rats. After successful anesthesia, blood was collected from the abdominal aorta. The collected blood was allowed to stand for 30 minutes and then centrifuged at 4000 rpm for 15 minutes. The supernatant was carefully aspirated to obtain serum. The ELISA kit was used to detect the levels of VCAM-1 and ICAM-1 in rat serum, and the corresponding biochemical index levels were calculated according to the instructions; HE staining: The dried sections were dewaxed with xylene and ethanol, and then stained in the order of hematoxylin for 5 min, 0.1% hydrochloric acid alcohol for 10 s, pure water washing 3 times, 0.6% ammonia water back to blue for 3 s, running water washing, and eosin staining for 3 min; after dehydration and dewaxing, the pathological microstructure of the thoracic aorta was observed under an optical microscope.

[0025] In vitro experiments: SD rats were adaptively fed for one week and randomly divided into a normal group and a qi-invigorating and blood-activating group, with 3 rats in each group. The qi-invigorating and blood-activating group was given oral administration with the amount of crude drug converted to 10.8 g / kg. The blank group was given an equal amount of normal saline by oral administration every day. Rats in each group were gavaged once a day for 7 consecutive days. One hour after the last day of administration, the rats were anesthetized by intraperitoneal injection of 3% pentobarbital, the abdominal hair of the rats was removed, and the abdomen was wiped with 75% alcohol cotton balls to collect blood from the abdominal aorta. The blood was centrifuged at 3000 rpm for 15 min, and the upper serum was carefully aspirated, inactivated at 56℃ in a water bath for 30 min, filtered with a 0.22 μm filter membrane, aseptically packaged in a biological safety cabinet and stored in a -20℃ refrigerator.

[0026] After establishing the high-glucose-induced HUVEC cell injury model, the cells were resuspended in high-glucose DMEM medium containing 10% FBS and diluted to a cell suspension with a density of 2×104 cells / mL. 100 μL of cell suspension was added to each well of a 96-well plate, and the 96-well plate was gently tapped to evenly distribute the cells in each well. The 96-well plate was then placed in a cell constant temperature incubator overnight until the cells adhered to the wall. After confirming that the cells adhered to the wall, the culture medium was discarded and the cells were divided into a normal group, a model group, and a drug-containing serum group with concentrations of 2.5%, 5%, 7.5%, and 10%, respectively, with 6 replicates in each group. 100 μL of high-glucose DMEM medium was added to each group of the normal group, and 100 μL of DMEM medium with a glucose concentration of 50 mmol / L was added to each well of the model group and the drug-containing serum group. After adding, the plates were placed in an incubator and incubated for 48 h. After the culture was completed, the culture medium in the wells was discarded, and 100 μL of high-glucose DMEM culture medium containing 10% blank rat serum was added to the normal group and model group, and high-glucose DMEM culture medium containing different concentrations of rat drug-containing serum was added to the drug-containing serum group. A blank well was set to zero (100 μL of cell-free culture medium was added), and the cells were placed in the incubator for 48 h. After the culture was completed, 50 μL of MTT reagent (2 mg / mL) was added to each well in the dark, and the cells were placed in the incubator for 4 h. After the incubation was completed, the liquid in the well was discarded and gently tapped on a clean toilet paper to completely absorb the liquid in the well. 150 μL of DMSO reagent was added to each well and placed on an oscillator for 5-10 min. After the cell crystals were completely dissolved, they were placed in an ELISA reader and the absorbance value was measured at a wavelength of 490 nm. The relative survival rate of cells in each group was calculated using the formula: relative cell survival rate = (OD490 high-glucose group-OD490 blank group) / (OD490 normal group-OD490 blank group).

[0027] The results were then characterized through cell scratch assay, potential target search, common target collection, and pathway enrichment analysis. The in vivo and in vitro experimental results are as follows.

[0028] In vivo experiments:

[0029] (I) General condition of rats: During the whole experiment, the rats in the normal group were in good condition, with quick reactions, active mental state, and smooth and shiny hair. The rats in the model group showed slow reactions, slow movements, and a depressed mental state as time went on. Their hair became increasingly dry and yellow. The Qi-invigorating and blood-activating group and the positive drug group showed some improvement. The rats in both groups were more active mentally, with smooth hair and more agile movements.

[0030] (II) Effect of Yiqi Huoxue prescription on rat body weight: As shown in Figure 1, the body weight of rats in the normal group continued to increase, and there was a significant difference between the normal group and the model group (P<0.01). The Yiqi Huoxue group and the positive drug group showed a difference from the model group at the 4th week, and there was a statistically significant difference (P<0.05).

[0031] (III) Effect of Yiqi Huoxue prescription group on FBG of rats: As shown in Figure 2, compared with the normal group, the FBG of the model group rats increased significantly during the 4-week period (P<0.01). In the 4th week, the FBG of the Yiqi Huoxue group and the positive drug group decreased significantly, and there was a statistical difference (P<0.05).

[0032] (IV) Levels of ICAM-1 and VCAM-1 in rats of each group: As shown in Figure 3, after 4 weeks of intervention, we detected the levels of serum ICAM-1 and VCAM-1 in rats of each group. The results showed that compared with the normal group, the levels of ICAM-1 and VCAM-1 in the model group were significantly increased (P<0.05) or (P<0.01), while the Yiqi Huoxue prescription could significantly reduce the levels of the two (P<0.01), which had the same effect as the positive drug group.

[0033] (V) Comparison of pathological sections of thoracic aorta of rats in each group: Figure 4 As shown in the figure, the phenotype of aortic endothelial cells in the normal group was normal, the endothelial layer thickness was moderate, the intima border was smooth and clear, and the endothelial cells were neatly arranged, while the phenotype of endothelial cells in the model rats was abnormal, the endothelial layer thickness increased, the intima was rough and disordered, and the endothelial cells were missing or irregular in shape, indicating that diabetes caused endothelial cell damage. After four weeks of treatment, the aortic intima of the Yiqi Huoxue group and the positive drug group was smooth and complete, the endothelial cells were arranged in a more orderly manner, and the histological morphology was significantly improved.

[0034] The results of in vivo experiments preliminarily found that the Yiqi Huoxue recipe can improve the blood sugar of diabetic rats, and has a certain improvement effect on the vascular damage caused by diabetes, and can delay the development of diabetic vascular diseases.

[0035] In vitro experiments:

[0036] (I) Effect of high glucose on HUVEC: As shown in Figure 5, when HUVEC cells were treated with high glucose for 24 hours, there was no significant difference in cell survival rate between the high glucose group and the normal group (P>0.05). When HUVEC cells were treated with high glucose for 48 hours, the cell survival rate of the high glucose group was significantly reduced compared with the normal group, with statistical difference (P<0.01).

[0037] (II) Determination of drug-containing serum concentration: As shown in Figure 6. MTT results showed that when the drug-containing serum concentration was 7.5%, the relative cell survival rate was the highest, and there was a significant difference compared with the high glucose group (P<0.05). In subsequent experiments, the drug-containing serum group with a concentration of 7.5% will be used as the treatment group.

[0038] (III) Effect of drug-containing serum on HUVEC cell migration ability: As shown in Figure 7, after high glucose induction, the healing ability of HUVEC was significantly reduced (P<0.01). After intervention with drug-containing serum, the healing ability of HUVEC was improved, and there was a very significant difference (P<0.01).

[0039] (IV) Screening of chemical components of Yiqi Huoxue prescription and acquisition of common targets for drugs and diseases: As shown in Figure 8, the chemical components of Yiqi Huoxue prescription were screened by SwissAMDE and analyzed by Swiss Target Prediction, and 196 components and 973 drug targets were obtained. 10048 related genes were retrieved from the GeneCards database, and 1004 highly related gene targets were screened. 778 related human gene targets were collected by Gene search in the NCBI database. 145 common targets for drugs and diseases were obtained through the R language "ggvenn" package and visualized as a Venn diagram.

[0040] (V) Target function and pathway enrichment analysis: 1. Protein interaction (PPI) network construction: As shown in Figure 9, 117 high-confidence targets were obtained using STRING and visualized as a PPI network diagram using Cytoscape 3.7.1 software.

[0041] 2. GO enrichment analysis: As shown in Figure 10, GO function enrichment of 117 high-confidence targets resulted in 3921 biological processes, 230 molecular functions, 90 cellular components, and a total of 4241 functional processes. These mainly involved biological processes related to wound healing and oxidative stress, such as wound healing, response to oxidative stress, and cellular response to chemical stress; cellular components related to cell membrane structures, such as membrane raft, membrane microdomain, and vesicle lumen; and molecular components related to enzymes and enzyme activities related to cell membrane construction, such as phosphatase binding, transmembrane receptor protein kinase activity, and transmembrane receptor protein tyrosine kinase activity. It can be seen that the Yiqi Huoxue prescription in the treatment of diabetic microvascular disease may be related to wound healing, oxidative stress and the composition of cell membrane structure.

[0042] 3. KEGG enrichment analysis: As shown in Figure 11, 117 high-confidence targets were obtained through KEGG pathway enrichment, involving a total of 246 significantly correlated pathways (Q value < 0.05), mainly involving the AGE-RAGE signaling pathway of diabetic complications, HIF-1, PI3K-Akt, MAPK and other pathways closely related to diabetic vascular diseases. Fig.11It is a bubble chart of KEGG enrichment analysis, involving the top ten diabetes-related pathways, including TNF signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, Lipid andatherosclerosis, Insulin resistance, HIF-1signaling pathway, Fluid shear stress andatherosclerosis, EGFR tyrosine kinase inhibitorresistance, Diabetic cardiomyopathy, and AGE-RAGE signaling pathway in diabetic complications.

[0043] (VI) Construction of the “TCM-ingredient-target-pathway-disease” network diagram: As shown in Figure 12, the active ingredients, targets and the top 20 KEGG enriched pathways of the Yiqi Huoxue recipe were screened and imported into Cytoscape3.7.1 to construct the “drug-ingredient-target-pathway-disease” network diagram. Fig.12 In the figure, A (dark purple) is the Yiqi Huoxue recipe, B (light purple) is the five Chinese medicines for nourishing yin in the Yiqi Huoxue recipe, C (blue) is the main related drug ingredients of the Yiqi Huoxue recipe (displayed as the corresponding ID of the HERB website), D (yellow) is the target related to drugs and diseases, E (light red) is the top 20 drug treatment-related pathways (displayed as pathway code IDs), and F (red) is the English name of the disease. Fig.12 From ABCDEF, we comprehensively show that the Yiqi Huoxue recipe uses five traditional Chinese medicines to act on multiple targets through multiple components and synergistically prevent and treat diabetic vascular diseases through multiple pathways; based on the results of KEGG enrichment analysis and the "drug-ingredient-target-pathway-disease" network diagram, we found that the MAPK signaling pathway plays a major role in the Yiqi Huoxue recipe's intervention in diabetic vascular diseases. Fig.11 As shown in Figure 2, the MAPK signaling pathway is highly enriched in KEGG enrichment analysis, and Fig.12It was also shown that proteins in the MAPK signaling pathway were closely related to the Yiqi Huoxue Recipe and diabetic vascular disease. Therefore, three key proteins in the MAPK signaling pathway, JNK, ERK, and p38, were screened for further verification.

[0044] (VII) Effect of Yiqi Huoxue Fang on the expression of JNK, ERK, p38 and p-JNK, p-ERK, p-p38 proteins: As shown in Figure 13, the expression of JNK, p38, and ERK in each group of cells was only slightly different, but there was no statistical difference (P>0.05). In terms of phosphorylation, there were significant differences in p-JNK, p-ERK, and p-p38 between the high glucose group and the normal group (P<0.01); the drug-containing serum group could reduce the expression of these proteins, and there was a significant difference (P<0.05 or P<0.01).

[0045] From the results of in vitro experiments, it can be concluded that the serum containing the Yiqi Huoxue recipe can improve the activity and migration ability of HUVEC induced by high glucose, and affect the proliferation and adhesion of HUVEC through the MAPK signaling pathway.

[0046] Based on the results of in vitro and in vivo experiments, the serum containing Yiqi Huoxue prescription can enhance the viability of HUVEC cells induced by high glucose and restore their migration ability, and can reduce the phosphorylation levels of three key proteins in the MAPK pathway. This proves that the treatment of diabetic vascular complications by Yiqi Huoxue prescription is likely related to the MAPK signaling pathway, which is of great significance for improving diabetic vascular complications.

Claims

1. A Qi-invigorating and blood-activating prescription for improving diabetic vascular complications, characterized in that: The prescription for invigorating qi and activating blood circulation is made up of astragalus, raw rehmannia, kudzu root, chuanxiong and radix trichosanthis.

2. A Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to claim 1, characterized in that: The Yiqi Huoxue recipe includes the following components: Preparation of the Qi-invigorating and Blood-activating Recipe: Weigh appropriate amounts of astragalus, raw rehmannia, kudzu root, chuanxiong, and trichosanthes root into a flask, add water to cover the mixture by 3 cm, and soak for 1-2 hours. After soaking, decoct twice, filter and rotary evaporate to concentrate to 0.85-1.20 g / mL, and freeze for later use; the amount of medicinal materials is 20-30 g of astragalus, 5-15 g of raw rehmannia and kudzu root, and 3-8 g of chuanxiong and trichosanthes root.

3. The Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to claim 1, characterized in that: The effect verification steps of the Yiqi Huoxue recipe are as follows: (1) Preparation of other related reagents: Metformin solution, 3% pentobarbital, streptozotocin (STZ) solution required for in vivo experiments, and 3% pentobarbital solution, electrophoresis buffer, transfer buffer, TBST solution, blocking buffer, cell lysis buffer, BCA protein quantitative colorimetric solution, and DMEM culture medium required for in vitro experiments. (2) In vitro and in vivo experiments: In vivo experiment: SPF male SD rats were selected, 5-10 rats were randomly selected as the normal group, and the rest of the rats were injected with streptozotocin STZ once by tail vein injection to induce T1DM rat model; after the model was successfully established, the rats were randomly divided into model group, positive drug group (metformin group), and Qi-invigorating and blood-activating group (Qi-invigorating and blood-activating prescription group), with 8 rats in each group, and the drug was administered for 4 weeks; the rats were weighed once a week and fasting blood glucose FBG was measured once; after 4 weeks of administration, the rats were anesthetized, serum was collected, and the levels of ICAM-1 and VCAM-1 in each group of rats were detected by ELISA; the aortic arch of the rats was collected and stored in 4% paraformaldehyde, and HE staining was performed to observe its histopathological changes; In vitro experiments: After human umbilical vein endothelial cells (HUVECs) were resuscitated, they were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), and models were established using DMEM medium with a glucose concentration of 50 mmol / L. After successful model establishment, the MTT method was used to detect the effects of different concentrations of drug-containing serum on cell viability to select the optimal drug-containing serum concentration. A cell scratch experiment was performed to detect the effects of drug-containing serum on high-glucose-induced HUVECs. Network pharmacology was used to analyze the targets of the Yiqi Huoxue recipe on diabetic vascular damage, and the Western blot (WB) method was used in vitro to verify its mechanism. (3) Data processing and result analysis: Analyze the test results of (2).

4. A Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to claim 3, characterized in that: Preparation of 50 mmol / L DMEM medium: Add anhydrous glucose powder to the cell and make up to 1 mL of PBS solution to prepare a glucose solution with a concentration of 3 mmol / L. Filter the glucose solution using a microporous filter membrane (0.22 μm), and then add the glucose solution to 50 mL of high-glucose DMEM medium to obtain 50 mmol / L DMEM medium, which is stored in a 4°C refrigerator.

5. The Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to claim 1, characterized in that: (2) Establishment of in vivo experimental diabetic rat model: Several SD rats were selected, with a body weight range of 260-280g and an age of 6-8 weeks. They were allowed to eat and drink freely during the feeding period. After 2 weeks of adaptive feeding, the rats were fasted but not watered for 14 hours. The fasting blood glucose and body weight at this time were tested and recorded. Streptozotocin (STZ) was used to establish the model. According to the rats' body weight, a single tail vein injection of STZ at 55 mg / kg was given. The rats continued to fast for 2 hours after the injection. After the injection of STZ, fasting blood glucose was tested on the 3rd, 7th and 10th days respectively. Rats with blood glucose greater than 16.7 mmol / L were selected as stable models for the next step of the experiment. (2) The process of establishing the high-glucose-induced HUVEC cell injury model described in the in vitro experiment is as follows: HUVECs in the culture dish are revived, cryopreserved after subculturing, and the cells are resuspended in high-glucose DMEM medium containing 10% FBS and diluted to a density of 3×10 4 / mL of cell suspension; add 100 μL of cell suspension to each well of a 96-well plate, tap the plate to evenly distribute the cells; place the plate in a cell culture incubator and culture overnight to allow the cells to adhere to the wall; after ensuring adhesion, discard the culture medium, add culture medium with different glucose concentrations, and place the plate in an incubator for culture for 24-48 h.

6. A Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to claim 5, characterized in that: (2) In vivo experimental index detection and HE staining thoracic aorta tissue morphology observation; the index detection includes general physical sign observation, weight recording, blood sugar detection and ecological index detection; the HE staining thoracic aorta tissue morphology observation process is to take the rat heart and aortic arch part, peel off the surrounding connective tissue, fix it in 4% paraformaldehyde for 24 hours, dehydrate and make it transparent, and then slice it according to the sequence of hematoxylin for 5 minutes, 0.1% hydrochloric acid alcohol for 10 seconds, pure water washing 2-5 times, 0.6% ammonia water blueing for 2-5 seconds, running water washing, eosin staining solution for 3 minutes, and then dehydrate the stained slices, seal them and observe them under an optical microscope.

7. The Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to claim 3, characterized in that: (2) The in vitro MTT method is as follows: after the culture is completed, 50 μL of MTT reagent (2 mg / mL) is added to each well under light-proof conditions, and the well is placed in the incubator for incubation for 4 h; after the incubation is completed, the liquid in the well is discarded and the well is gently tapped on clean toilet paper to completely absorb the liquid in the well; 150 μL of DMSO reagent is added to each well and the well is placed on an oscillator for 5-10 min; after the cell crystals are completely dissolved, the well is placed in an ELISA reader and the absorbance value is measured at a wavelength of 490 nm to calculate the relative survival rate of each group of cells. The formula is: relative cell survival rate = (OD490 high glucose group - OD490 blank group) / (OD490 normal group - OD490 blank group); (2) The process of the cell scratch test is as follows: resuspend the cells in high-glucose DMEM medium containing 10% FBS and dilute to a density of 3×10 4 / mL cell suspension; add 2mL of cell suspension to each well of a 96-well plate, tap the well plate to evenly distribute the cells; place the well plate in a cell culture incubator and culture overnight to allow the cells to adhere to the wall; after confirming the adhesion, discard the culture medium, add PBS to wash the residual culture medium, place it in an incubator and culture it for 24-48 hours, then add high-glucose DMEM culture medium containing 2.5% blank rat serum and 7.5% drug-containing serum and place it in an incubator for intervention for 48 hours, after which the liquid in the well is discarded and the well is scratched with a pipette tip, PBS is added, and then the excess cells are washed off; the cell scratches obtained are observed under an inverted microscope, and the cell migration is observed and photographed under an inverted microscope at 0 h, 6 h, 12 h, and 24 h after scratching, and the area difference between 0 h and 24 h is determined by Image J.

8. The Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to claim 7, characterized in that: The process of network pharmacology analysis is as follows: after screening the ingredients of the Yiqi Huoxue recipe, drug target prediction and disease target collection are performed; after obtaining the common targets of drugs and diseases, protein interaction PPI network construction and enrichment analysis are performed; finally, a "TCM-ingredient-target-pathway-disease" network is constructed based on the relationship between protein targets and KEGG signaling pathways.

9. The Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to claim 8, characterized in that: The process of Western blot detection of protein levels was to extract total protein from the cells after the intervention, determine the protein concentration using the BCA method and prepare protein samples, load the protein samples for electrophoresis and incubate with antibodies, and finally perform imaging and subsequent data analysis. All statistical analyses were performed using SPSS 22.0 software and GraphPad Prism8.4.3 software.

10. An application of a Qi-invigorating and blood-activating prescription for improving diabetic vascular complications, characterized in that ： A Qi-invigorating and blood-activating prescription for improving diabetic vascular complications according to any one of claims 1-9 is prepared and verified, and the Qi-invigorating and blood-activating prescription is applied to the treatment of diabetic vascular complications and the verification method is applied to other related pharmacological experiments, and the Qi-invigorating and blood-activating prescription is verified and characterized by constructing a pharmacological network of a model sample.

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