New application of compound high spot-nourishing tablet

Abstract

The invention discloses application of a compound high spot-nourishing tablet in preparation of a medicine for treating cerebral ischemia-reperfusion injury. A rat cerebral ischemia reperfusion injury model is established through middle cerebral artery embolism, and serum, pathology and infarction areas of rats are observed and detected. Results show that the neurological score of the model group ZeaLonga is significantly increased, pathological changes are large, proinflammatory factors TNF-alpha, IL-6 and IL-1beta are significantly increased, oxidation promoting MDA is significantly increased, and antioxidant SOD expression is significantly increased. And compared with a model group, the weight, the neurological score and the survival rate are obviously improved. The results of HE staining, TTC staining and TUNEL staining show that the nerve cell morphology, arrangement, infarct area and cell apoptosis condition of each administration group are obviously improved. The application provided by the invention can improve the behavior of rats, reduce the infarct area, protect nerve cells and reduce the apoptosis of ischemic regions; by reducing inflammation and oxidation in rat brain tissues, damage of inflammation and oxidative stress to the rat brain tissues is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to the technical field of traditional medicine, and more particularly to the application of the compound Gaozhi tablet in the preparation of a drug for cerebral ischemia-reperfusion injury. BACKGROUND

[0002] Cerebral ischemic reperfusion injury (CIRI) refers to the complex pathological process caused by the restoration of blood supply after the interruption of cerebral blood flow. This injury is very common in ischemic diseases such as ischemic stroke and cardiac arrest, leading to a high incidence and high disability rate, and is one of the important reasons for the death and disability of patients with acute ischemic stroke. In 2019, the prevalence of ischemic stroke in China was 1700 / 10 million (age-standardized prevalence 1256 / 10 million), and the prevalence of hemorrhagic stroke was 306 / 10 million (age-standardized prevalence 215 / 10 million). The prevalence of stroke in the population aged 40 and above in China increased from 1.89% in 2012 to 2.58% in 2019. In 2019, the number of people with and without stroke in the population aged 40 and above in China was about 1704 million. With the popularization of thrombolysis and endovascular thrombectomy for acute stroke, more and more patients can restore blood supply to the ischemic area in time, and the efficacy is improved. However, it is also inevitable to have neurological dysfunction caused by prolonged ischemic time, mainly including limb weakness, sensory abnormalities, language disorders (aphasia), visual impairment and other symptoms, which seriously affect the quality of life of patients. There are many methods for treating cerebral ischemia-reperfusion, including drug therapy such as antioxidant vitamin C, excitatory amino acid antagonist NMDAR, and soy isoflavones targeting iron death. Traditional Chinese medicine and electro-acupuncture and moxibustion are also believed to inhibit the production of reactive oxygen species (ROS) and iron death in cerebral ischemia-reperfusion injury. However, the current treatment methods only target a certain pathological link of cerebral ischemia-reperfusion, and due to the complexity of the pathological mechanism of CIRI, single-target drugs often have difficulty achieving good results. Traditional Chinese medicine has the characteristics of multiple components and multiple targets, and can treat CIRI from multiple angles such as antioxidant, anti-inflammatory, regulation of neural plasticity, and maintenance of blood-brain barrier stability. SUMMARY

[0003] Based on the fact that the compound Gaozhi tablet in the prior art is mainly used for the treatment of mental diseases such as depression and senile mental diseases with sleep disorders, the present application provides a new use of the compound Gaozhi tablet, and the application of the compound Gaozhi tablet in the preparation of a drug for cerebral ischemia-reperfusion injury, which can effectively improve the abnormal behavior, nerve cell necrosis, apoptosis, inflammation and oxidative stress caused by cerebral ischemia-reperfusion injury.

[0004] In order to achieve the above technical effects, the application is realized by the following technical solutions.

[0005] The application of the compound Gaozhi tablet in the preparation of a drug for cerebral ischemia-reperfusion injury is provided.

[0006] The dose of the compound Gaozhi tablet ranges from 0.0135 g / ml to 0.054 g / ml.

[0007] The application is at least one of improving the injury caused by cerebral ischemia-reperfusion or reducing the infarction area.

[0008] The application is at least one of significantly reducing the necrosis, atrophy and necrosis of nerve cells, and effectively improving the morphology and integrity of nerve cells in the brain tissue of a cerebral ischemia-reperfusion injury rat.

[0009] The application is significantly reducing the apoptosis cells in the brain tissue of a cerebral ischemia-reperfusion injury rat.

[0010] The application is improving the expression of key genes in cerebral ischemia-reperfusion injury.

[0011] The application is at least one of significantly reducing the expression of pro-inflammatory factors TNF-ɑ, IL-6 and IL-1β, and pro-oxidant MDA in the brain tissue of a cerebral ischemia-reperfusion injury, and significantly increasing SOD in the brain tissue.

[0012] The application of the compound Gaozhi tablet in the preparation of a drug for cerebral ischemia-reperfusion injury is provided, which is prepared from the following raw materials: bull tongue grass, European car chen root, sandalwood, perilla fruit, large leaf bloodwort, bluegrass, home alone, lavender, coriander seed, bull tongue flower and cocoon medicinal materials, and pharmaceutically acceptable excipients are added. According to the theory of traditional Chinese medicine and the characteristics of national medicine, a simple and reasonable preparation method is adopted, so that the effective components of each medicinal material for treating cerebral ischemia-reperfusion injury can play a pharmacodynamic effect. The compound medicine has definite curative effect, less side effect and low recurrence rate, and is suitable for preparation into tablets, providing convenience for the application of patients with cerebral ischemia-reperfusion injury.

[0013] The compound Gaozhi tablet has the characteristics of unique prescription, definite curative effect, less side effect, multiple effects and multiple targets, and has the effects of strengthening heart and brain, calming the nerves, dredging the vessels, relieving insomnia, headache and neurasthenia, and treating cerebral ischemia-reperfusion injury, and is suitable for oral administration for patients with cerebral ischemic injury.

[0014] The compound Gaozhi tablet has the following pharmacological characteristics: Anchusa italica (L.) Moench is the dry aboveground part of Anchusa italica (L.) Moench or Borago officinalis L. in Boraginaceae, also known as Gaosiwan or Gaosiban. It is a common medicinal material in many classic Uyghur medicine prescriptions. It is believed in Uyghur medicine that Anchusa italica (L.) Moench has the effects of removing dampness and heat, moistening the lung and nourishing the brain, dispelling cold and tonifying the heart, and refreshing the heart and improving the spirit. Studies have shown that Anchusa italica (L.) Moench can act on cerebral ischemia-reperfusion injury by reducing inflammatory factors such as interleukin-1β, interleukin-6, and tumor necrosis factor-α.

[0015] Lavandula angustifolia Mill is a shrub of the genus Lavandula in the Lamiaceae family. Its main chemical components are volatile oil and flavonoids, and it has anti-inflammatory, antibacterial, antioxidant, and central nervous system effects. Volatile oil plays an important role in the treatment of cerebral ischemia-reperfusion injury by inhibiting major pro-inflammatory factors and their receptors, balancing inflammatory signals, and thus effectively improving cerebral ischemia-reperfusion injury by changing the inflammatory microenvironment.

[0016] Dracocephalum moldavica L. is a traditional medicinal material for the Uyghur and Mongolian nationalities, belonging to the Lamiaceae family. It has the effects of tonifying the heart and brain, promoting blood circulation and resolving phlegm, diuresis, and relieving cough. Its main components include flavonoids, steroids, volatile oils, and various trace elements. Among them, flavonoids have been found to regulate intracellular and extracellular signaling pathways, regulate autophagy and apoptosis, and have the effects of anti-myocardial ischemia and anti-atherosclerosis. Recently, researchers have analyzed 56 monomer components of Dracocephalum moldavica L. total flavonoids, which may improve cerebral ischemia-reperfusion injury through mechanisms such as antioxidant stress, regulation of autophagy and apoptosis, and promotion of blood vessel regeneration.

[0017] Perilla frutescens (L.) Britt is the fruit of Perilla frutescens (L.) Britt in the Lamiaceae family. Its main components include phenolic acids, fatty acids, flavonoids, and volatile oils. Modern pharmacological studies have shown that it mainly has the effects of lowering lipid, relieving cough and asthma, anti-inflammatory, antioxidant, and enhancing memory. Studies have found that Perilla frutescens (L.) Britt extract can reduce mouse colon pathology by inhibiting inflammatory factors such as tumor necrosis factor-α, interleukin-6, and interleukin-1β. Therefore, Perilla frutescens (L.) Britt may also improve cerebral ischemia-reperfusion injury by inhibiting inflammatory factor levels.

[0018] Limonium gmelinii (Willd.) Kuntze is a perennial herb of the Glinus genus in the Plumbaginaceae family, which is mainly used for the treatment of cervical erosion, endometritis, and other inflammatory diseases, as well as hemostasis for uterine bleeding and hematuria. Studies have found that Limonium gmelinii (Willd.) Kuntze total flavonoids have certain inhibitory effects on the production of hydroxyl free radicals, superoxide anion free radicals, and lipid peroxidation, and can play a role in treating cerebral ischemia-reperfusion injury by inhibiting oxidative stress.

[0019] Sandalwood, derived from the sandal tree, is a precious spice and medicinal material. Its main chemical components include alpha-santalol and beta-santalol, which give sandalwood its unique aroma and pharmacological activity. In traditional medicine, sandalwood is used to treat various diseases such as indigestion, respiratory tract infections, and skin problems. In addition, sandalwood has a calming and anti-inflammatory effect, often used to relieve anxiety and stress. In recent years, research has found that sandalwood has potential protective effects on cerebral ischemia-reperfusion injury. Cerebral ischemia-reperfusion injury refers to further tissue damage caused by oxidative stress and inflammatory response after the restoration of blood flow after interruption of blood supply to the brain. The active ingredients in sandalwood can reduce this damage through antioxidant and anti-inflammatory mechanisms. Specifically, sandalol can inhibit the generation of free radicals, reduce cell apoptosis, and protect nerve cells from ischemia-reperfusion-induced damage. These findings provide scientific evidence for the application of sandalwood in the field of neuroprotection, demonstrating its potential in modern medicine.

[0020] The implementation of the above specific technical solutions provided by the present application can achieve the following beneficial effects: The application provides the use of the compound Gaozibo tablets in the preparation of a drug for cerebral ischemia-reperfusion injury. The rat model of cerebral ischemia-reperfusion injury is established by middle cerebral artery occlusion, and the rat serum, pathology, and infarct area are observed and detected. The results show that compared with the sham operation group, the ZeaLonga neurological score of the model group is significantly increased, the pathological changes are large, the inflammatory factors (TNF-ɑ, IL-6 and IL-1β) are significantly increased, and the pro-oxidative MDA is significantly increased, indicating that the modeling is successful. Compared with the model group, the body weight, neurological score and survival rate are significantly improved. The HE staining, TTC staining and TUNEL staining results show that the morphology, arrangement, infarct area and apoptosis of nerve cells in each drug administration group are significantly improved. The expression of pro-inflammatory factors TNF-ɑ, IL-6 and IL-1β and pro-oxidative MDA in brain tissue is significantly reduced, and the expression of antioxidant SOD is significantly increased. The drug administration groups of the present application can improve the behavior of rats, reduce the infarct area, protect nerve cells, and reduce apoptosis in the ischemic area; by reducing inflammation and oxidation in the brain tissue of rats, the damage of inflammation and oxidative stress to the brain tissue of rats is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The survival of rats in each group is shown in the graph.

[0022] Figure 2 The ZeaLonga neurological score changes of rats in each group before and after administration are shown in the graph.

[0023] Figure 3 The body weight changes of rats in each group for 14 days are shown in the graph.

[0024] Figure 4The TTC staining of each group is shown.

[0025] Figure 5 The HE staining of each group of mice is shown.

[0026] Figure 6 The TUNEL staining of each group of mice is shown.

[0027] Figure 7 The expression levels of TNF-ɑ, IL-6 and IL-1β, MDA and SOD in the serum of each group are shown. DETAILED DESCRIPTION

[0028] The application will be described in greater detail below with reference to the following Examples, but the application is not limited to the following Examples. All the raw and auxiliary materials selected in the application are well known in the art, and the % involved in the application is mass percentage, unless otherwise specified.

[0029] The related reagents used in the application are: Compound Gaozibo Tablets (Xinjiang Uygur Pharmaceutical Industry, batch number: ); Atorvastatin (Hui Zhi Pharmaceutical (Dalian) Co., Ltd., batch number: 8190146); General tissue fixative (Biosharp Technology Co., Ltd., batch number: BL539A); Tumor necrosis factor-ɑ assay kit (Wuhan Kelu Biological Technology Co., Ltd., batch number: G1076); Malondialdehyde assay kit (Nanjing Jiancheng Biological Engineering Institute, batch number A003-1-2); Interleukin 6 assay kit (Wuhan Kelu Biological Technology Co., Ltd., batch number E-BC-K031-M); Interleukin 1β assay kit (Wuhan Kelu Biological Technology Co., Ltd., batch number: E-BC-K020-M); Superoxide dismutase assay kit (Wuhan Elirete Biological Technology Co., Ltd., batch number A015-2-1).

[0030] The related instruments used in the application are: MCAO thread (Mayue Biotechnology), dehydrator (DIAPATH); microplate reader (Thermo Fisher Scientific (Shanghai) Instrument Co., Ltd.); large-capacity high-speed bench centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); upright white light photographing microscope (Olympus); vortex mixer (Shanghai Luxi Analytical Instrument Factory Co., Ltd.); decolorization shaker (Beijing Liuyi Instrument Factory).

[0031] In order to better explain the application, the main content of the application will be further illustrated in combination with specific examples below, but the content of the application is not limited to the following examples only. If not specifically indicated, the technical means used in the examples is the conventional means well known to those skilled in the art, and the raw materials used are commercially available goods.

[0032] Example 1: Application of Compound Gaozibo Tablets in the preparation of drugs for cerebral ischemia-reperfusion injury The application aims to provide an application of compound Gaotiao tablets in preparation of a medicine for cerebral ischemia-reperfusion injury.

[0033] The dose of the compound Gaotiao tablets ranges from 0.0135 g / ml to 0.054 g / ml.

[0034] The application is at least one of improving the injury caused by cerebral ischemia-reperfusion or reducing the infarction area.

[0035] The application is at least one of significantly reducing the necrosis, atrophy and necrosis of nerve cells, and effectively improving the morphology and integrity of nerve cells in the brain tissue of a cerebral ischemia-reperfusion injury rat.

[0036] The application is significantly reducing the apoptosis cells in the brain tissue of a cerebral ischemia-reperfusion injury rat.

[0037] The application is improving the expression of key genes in cerebral ischemia-reperfusion injury.

[0038] The application is at least one of significantly reducing the expression of pro-inflammatory factors TNF-ɑ, IL-6 and IL-1β, and pro-oxidative MDA in the brain tissue of a cerebral ischemia-reperfusion injury, and significantly increasing SOD in the brain tissue.

[0039] Example 2: Animal test 1. Materials and methods 1.1 Experimental animals SPF level SD mice, weighing 240±2g, male; all provided by the animal experiment center of Xinjiang Medical University, animal experiment production license number:, the feeding environment is (23±2) ℃, humidity (60±5) %, and the light is 12h / dark 12h. The nest is changed twice a week, and water and feed are added daily to meet the normal growth needs of the experimental mice. The animal experiment scheme in this study is approved by the experimental animal ethics committee of Xinjiang Medical University (ethics approval number: ), and all animal experiment operations comply with the 3R principle of experimental animals and the relevant experimental animal welfare ethics standards at home and abroad; 1.2 Preparation of experimental drugs Test drug: the compound medicine for treating cerebral ischemia-reperfusion injury described in the application, the adult daily dose is 0.0428g / kg, which is converted into the daily dose of rats as 0.27g / kg (medium dose), the concentration is 0.027g / ml; the low dose is (0.135g / kg), the concentration is 0.0135g / ml; the high dose is (0.54g / kg), the concentration is 0.054g / ml; the administration volume is 10ml / kg.

[0040] Positive control drug: atorvastatin, Hui Zhi Pharmaceutical (Dalian) Co., Ltd. (purchased from a pharmacy, purchase date: April 12, 2025); batch number: 8190146; specification: 20 mg / tablet, 7 tablets / plate.

[0041] Positive control drug preparation method: atorvastatin daily dose 0.286 mg / kg, converted to rat daily dose 1.8 mg / kg, concentration 0.18 mg / ml, administration volume 10 ml / kg.

[0042] 1.3 Method 1.3.1 Modeling After the rats were adaptively fed for one week, the operation was performed when the body weight reached the range of (240±20) g. The rats were fasted for 12 h and watered for 4 h before the operation. About 1 h before the operation, the rats should be familiar with the surrounding environment. The method for establishing the MCAO model was based on the previous laboratory research, and the ischemia-reperfusion model was established. Before the operation, the rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution (diluted with normal saline). During the operation, the rats were fixed in a supine position. After the skin of the neck was disinfected, an incision was made in the midline of the neck. The subcutaneous muscles were bluntly separated with forceps. The vagus nerve and blood vessels, including the common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA), were separated. Care should be taken not to injure the vagus nerve. The CCA and ICA were clamped with a vascular clamp. A surgical knot was tied on the proximal end of the ECA without tightening. The distal end of the ECA was ligated with black and red threads. The ECA was cut between the two threads. A V-shaped opening was cut on the ECA. The black thread was pulled to push the pre-prepared thread plug into the blood vessel through the V-shaped opening of the ECA. The vascular clamp on the ICA was loosened. The thread plug was inserted into the ICA at the bifurcation of the CCA. When the head end of the thread plug was about 18 mm away from the bifurcation, the blood flow into the brain was blocked. If the resistance was felt, the advancement should be stopped immediately, otherwise it would cause cerebral hemorrhage. The surgical knot on the ECA was tightened. The vascular clamp on the CCA was removed. The blood flow into the brain was blocked for 2 h. During this period, the opening was kept moist with a cotton ball soaked in normal saline. After 2 h of ischemia, the thread plug was removed. The opening of the ECA was tightened. The skin of the rat's neck was sutured. Iodine was used for disinfection. The rat's body temperature was maintained throughout the operation and after the operation.

[0043] 1.3.2 Administration: ① Sham operation group: only the main blood vessels in the neck were separated, without inserting the thread plug to block the blood supply to the brain. Distilled water was given daily. ② Model group: ischemia for 2 h, removal of the thread plug to restore blood supply, no drug administration, and sacrifice for sample collection after 14 days.

[0044] ③ Positive control group: After modeling, atorvastatin 1.8 mg / kg was given to the rats, once every 24 h, for a total of 14 times, and the rats were sacrificed on the 15th day. ④ Low-dose administration group: After modeling, compound Gaozhi tablets 0.135 g / kg were given to the rats, once every 24 h, for a total of 14 times, and the rats were sacrificed on the 15th day. ⑤ Medium-dose administration group: After modeling, compound Gaozhi tablets 0.27 g / kg were given to the rats, once every 24 h, for a total of 14 times, and the rats were sacrificed on the 15th day. ⑥ High-dose administration group: After modeling, compound Gaozhi tablets 0.54 g / kg were given to the rats, once every 24 h, for a total of 14 times, and the rats were sacrificed on the 15th day. 1.3.3 Detection index (1) Zea Longa neurological score, body weight change and survival curve After the rats were completely awake after the operation, the rats with a score of 1-3 were randomly assigned into groups according to the standard of Zea Longa scoring method, and the rest were excluded.

[0045] After the rats were completely awake after the operation, the rats with a score of 1-3 were randomly assigned into groups according to the standard of Zea Longa scoring method, and the rest were excluded. To judge the neurological impairment of rats in each treatment group, the scoring criteria were 0-4 points, and a total of five grades, the higher the score, the more serious the neurological impairment. The scoring criteria are shown in Table 1: Table 1: Zea Longa neurological scoring criteria

[0046] (2) TTC staining Tissue sampling: 1% sodium pentobarbital was injected intraperitoneally to anesthetize the rats, and after blood was taken from the abdominal aorta, the heart was perfused (a small hole was cut in the right atrial appendage, and perfusion was not less than 20 min, of which 50-100 mL of physiological saline was perfused), and then the brain tissue was immediately removed by decapitation and frozen in a-80℃ refrigerator for 5 min.

[0047] Slicing: The brain tissue was cut into 6 continuous coronal sections of 2 mm thickness in a brain section mold.

[0048] Staining: Soak in TTC solution and stain in a 37℃ incubator for 30 min, turn over every 5 min, and gently shake the culture dish.

[0049] Fixing: After staining is complete, fix in 4% paraformaldehyde fixing solution until the tissue is hardened, then take out and air dry, and place in order to collect images using a camera.

[0050] (3) HE staining After anesthesia, abdominal aorta blood, heart perfusion, cut a small mouth in the right atrium, perfusion for not less than 20 min, among them, saline perfusion 50~100mL, take the complete brain tissue, make wax block do coronal section, thickness 5μm. Xylene and anhydrous ethanol dewaxing, hematoxylin staining, 1% eosin reagent, using gradient ethanol dehydration, finally xylene transparent mounting. Under light microscope, observe the morphological characteristics of nerve cells in the cortex of rats in each group.

[0051] (4) TUNEL staining Take the proteinase K in the kit, dilute with PBS according to 1:9, prepare working solution, incubate in 37℃ water bath for 20 min, wash with PBS for 3 times, 5 min each time.

[0052] Prepare membrane breaking solution, incubate at room temperature for 10 min, wash with PBS for 3 times, 10 min each time.

[0053] Incubate with 3% H2O2 at room temperature for 15~30 min, block endogenous hydrogen peroxidase, wash with PBS for 3 times, 5 min each time.

[0054] Take TdT enzyme, Biotin-dUTP reaction solution and EB equilibration buffer in the kit, mix them in order according to 2:5:50, prepare appropriate amount of incubation solution, incubate at 37℃ for 1h~1.5h or overnight at 4℃ in dark.

[0055] Wash with PBS for 3 times, 5 min each time, add appropriate amount of Streptavidin-HRP reaction solution (dilute with PBS according to 1:200), incubate in 37℃ incubator for 30 min.

[0056] Wash with PBS for 3 times, 5 min each time, prepare DAB working solution, control color development under microscope, positive is brownish yellow, wash with tap water to stop color development.

[0057] (5) Effect of compound Gaodizhi tablet on serum inflammatory factors TNF-ɑ, IL-6 and IL-1β and oxidative stress malondialdehyde MDA and superoxide dismutase SOD in cerebral ischemia-reperfusion rats Take serum and tissue specimens: blood was naturally coagulated at room temperature for 2h, centrifuged at 3000r / min for 20min, and the supernatant was collected for standby.

[0058] Divide and add sample: divide the standard hole area and sample hole area on the coated plate, add standard sample with different concentration gradient to each standard hole, 100µl. Establish blank hole without any reagent. Establish sample hole and add 100µl of each group sample, shake gently.

[0059] Add enzyme incubation: add 100µl of enzyme-labeled reagent to each hole, seal the plate with sealing film, and incubate at 37℃ for 80min.

[0060] Wash the coated plate: remove the sealing film, discard the liquid and try to pat dry, add diluent to each well and stand for 1 min, try to completely pour the liquid, and pat dry the coated plate after repeated washing for 5 times.

[0061] Color development: first add color developer A liquid, 90 μl per well, mix gently, seal the plate with a sealing plate, and incubate at 37°C for 20 min in the dark.

[0062] Determination: add 50 μl of termination liquid to terminate the reaction, select 450 nm wavelength, and measure the absorbance od value of each well.

[0063] 1.4 Statistical processing: The data measurement results are expressed by ±s, the paired t test is used between two groups, the ordinary one-way analysis of variance (ANOVA) is used between multiple groups, and P<0.05 is statistically significant.

[0064] 2. Results 2.1 Zea Longa neurological function score, body weight change and survival curve Referring to the accompanying Figure 1 and Table 2, no death occurred in the sham operation group after the operation; compared with the sham operation group, 4 rats in the model group died at 1, 3 and 6 days after the operation; 3 rats in the low-dose group died at 1, 4 and 6 days after the operation; 3 rats in the medium-dose group died at 2 and 5 days after the operation; and 1 rat in the high-dose and atorvastatin groups died at 1 day after the operation; referring to the accompanying Figure 2 and Table 3, compared with the sham operation group, the Zea Long scores of the model group, the low-dose group, the medium-dose group and the high-dose group were significantly increased (P<0.001), which was statistically significant, indicating that the modeling was successful; referring to the accompanying Figure 3 , the body weight of the sham operation group increased after the operation, and compared with the sham operation group, the body weight of the model group decreased significantly after the operation, and continuously decreased to (190±10) g at the 3rd day after the operation; the body weight of the low-dose group, the medium-dose group and the high-dose group decreased significantly at the 3rd day after the operation, and recovered to the preoperative weight at the 4th day; compared with the model group, the sham operation group, the low-dose group, the medium-dose group and the high-dose group had statistical significance. The present application significantly improves the survival rate of rats with cerebral ischemia-reperfusion injury, and the body weight of the compound high-zi tablet group is significantly increased compared with the model group.

[0065] Table 2: Survival of rats in each group (n=12)

[0066] Table 3: Changes in Zea Longa neurological function scores of rats in each group before and after administration (±s, n=6) ​​

[0067] Note: Compared with the normal group, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0068] 2.2 TTC staining TTC staining was used to observe the infarct area in rat brain tissue. (See Appendix) Figure 4 As shown, the results indicated that the brain tissue of the sham-operated group was stained red with TTC staining and no infarct area was observed. Compared with the sham-operated group, the model group showed a large area of ​​infarct. Compared with the model group, the low-dose group of Compound Gao Zi Ban Tablets did not show significant improvement in the infarct area. The medium and high doses of Compound Gao Zi Ban Tablets showed significant improvement in the infarct area. The results indicate that the present invention can improve the damage caused by cerebral ischemia-reperfusion and reduce the infarct area.

[0069] 2.3 HE staining See appendix Figure 5 The results showed that in the sham-operated group, the neurons in the cortical brain tissue of rats were round, neatly arranged, with clear nucleoli and uniform chromatin distribution. Compared with the sham-operated group, the neurons in the model group were damaged, atrophied, and even necrotic, with disordered arrangement, blurred nucleoli, uneven chromatin distribution, and pale staining. Compared with the model group, the low-dose group had no significant therapeutic effect. The neurons in the medium- and high-dose groups were loose, relatively neatly arranged, with relatively clear nucleoli, occasionally showing atrophy and necrosis, and with relatively uniform chromatin distribution. This indicates that the present invention significantly reduces neuronal necrosis, atrophy, and death, and can effectively improve the morphology and integrity of neurons in the brain tissue of rats with cerebral ischemia-reperfusion injury.

[0070] 2.4 TUNEL staining As shown in Table 4 and Appendix Figure 6 The results showed that, compared with the sham-operated group, a significant increase in the number of apoptotic cells was observed in the brain tissue of rats in the model group. Compared with the model group, the number of apoptotic cells in the low, medium and high dose groups of Compound Gao Zi Ban Tablets showed a decreasing trend, with statistical differences. This indicates that the present invention can significantly reduce apoptotic cells in the brain tissue of rats with cerebral ischemia-reperfusion injury.

[0071] Table 4: Apoptosis of rat brain tissue cells in each group stained with TUNEL ( ±s, n=3)

[0072] Note: Compared with the normal group, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0073] 2.5 Effects of Compound Gaoziban Tablets on Serum Pro-inflammatory Factors TNF-α, IL-6, and IL-1β, and Oxidative Stress Factors Malondialdehyde (MDA) and Superoxide Dismutase (SOD) in Rats with Cerebral Ischemia-Reperfusion Injury See Figure 7 The results showed that compared with the sham-operated group, the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the serum of rats in the model group was significantly increased (P < 0.001), which was statistically significant. Compared with the model group, the expression of pro-inflammatory factor TNF-α in the serum of rats in the low, medium, and high dose groups of Compound Gaoziban Tablets and the atorvastatin group was significantly decreased (P < 0.001), which was statistically significant. Compared with the model group, the expression of pro-inflammatory factor IL-6 in the serum of rats in the low, medium, and high dose groups of Compound Gaoziban Tablets and the atorvastatin group was significantly decreased (P < 0.001), which was statistically significant. Compared with the model group, the expression of pro-inflammatory factor IL-1β in the serum of rats in the low, medium, and high dose groups of Compound Gaoziban Tablets and the atorvastatin group was significantly increased. The levels of MDA in the serum of rats were significantly reduced, with statistically significant increases observed in the medium- and high-dose groups of Compound Gaoziban Tablets and the atorvastatin group. Compared with the sham-operated group, the serum MDA expression in the model group was significantly increased (P < 0.05). Conversely, compared with the model group, the serum MDA expression in the low-, medium-, and high-dose groups of Compound Gaoziban Tablets and the atorvastatin group was significantly decreased (P < 0.05). Compared with the sham-operated group, the serum SOD expression in the model group was significantly decreased (P < 0.001). Conversely, compared with the model group, the serum SOD expression in the low-, medium-, and high-dose groups of Compound Gaoziban Tablets and the atorvastatin group was significantly increased, with statistically significant increases observed in the low- and high-dose groups. These findings suggest that Compound Gaoziban Tablets can alleviate pro-inflammatory factors TNF-α, IL-6, IL-1β, and oxidative damage MDA in the brain tissue of rats with cerebral ischemia-perfusion injury, and increase SOD levels in brain tissue. This invention can effectively reduce the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β and pro-oxidative MDA in the brain tissue of rats with cerebral ischemia-reperfusion injury, significantly increase SOD in brain tissue, and significantly reduce cerebral ischemia-reperfusion injury in rats.

[0074] Table 5: Serum expression levels of TNF-α, IL-6, IL-1β, MDA, and SOD ( ±s, n=6)

[0075] Note: Compared with the normal group, ###P<0.001; compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0076] Cerebral infarction refers to ischemic and anoxic necrosis of local tissues caused by cerebral blood circulation disorder, and after taking thrombolysis and intravascular intervention to remove thrombus, cerebral ischemia-reperfusion injury occurs; cerebral ischemia-reperfusion injury refers to that after ischemic brain tissue is restored (reperfusion), nerve injury and cell death are aggravated. The mechanism of occurrence includes oxidative stress, inflammatory reaction, calcium overload and blood-brain barrier destruction; inflammation is the core mechanism of cerebral ischemia-reperfusion injury, involving activation of microglia, neutrophils, inflammatory factors (TNF-α, IL-1β) and NLRP3 inflammasome; inflammation aggravates blood-brain barrier destruction, neuron death and microcirculation disorder, resulting in secondary injury. Therefore, anti-inflammatory treatment can become an important strategy for brain protection.

[0077] The technical scheme provided by the application establishes a rat cerebral ischemia-reperfusion injury model through middle cerebral artery embolism, and observes and detects the serum, pathology and infarction area of the rat. The results show that compared with the sham operation group, the ZeaLonga neurological function score of the model group is significantly increased, the pathological changes are large, the inflammatory factors (TNF-ɑ, IL-6 and IL-1β) are significantly increased, and the pro-oxidative MDA is obviously increased, indicating that the modeling is successful. Compared with the model group, the body weight, neurological function score and survival rate are obviously improved. The HE staining, TTC staining and TUNEL staining results show that the morphology, arrangement, infarction area and apoptosis of nerve cells of each administration group are obviously improved. The expression of pro-inflammatory factors TNF-ɑ, IL-6 and IL-1β and pro-oxidative MDA in brain tissue is obviously reduced, and the expression of antioxidant SOD is obviously increased. The administration groups of the application can improve the behavior of rats, reduce the infarction area, protect nerve cells and reduce cell apoptosis in ischemic areas; by reducing inflammation and oxidation in the brain tissue of rats, the damage of inflammation and oxidative stress to the brain tissue of rats is reduced.

[0078] In summary, the application described herein is a compound drug for treating cerebral ischemia-reperfusion injury, which can effectively improve the abnormal behavior, nerve cell necrosis, cell apoptosis, inflammation and oxidative stress and other injuries caused by cerebral ischemia-reperfusion. According to the experimental results, the application can improve the damage caused by cerebral ischemia-reperfusion.

[0079] The above-described embodiments are only some of the embodiments of the application, not all the embodiments. The detailed description of the embodiments of the application is not intended to limit the scope of the claimed application, but only represents selected embodiments of the application. All other embodiments obtained by related deduction and replacement made by those skilled in the art under the condition of the concept of the application, without making creative efforts, belong to the scope of protection of the application.

Claims

1. Application of compound Gaozibo tablets in preparation of drugs for cerebral ischemia-reperfusion injury.

2. Use according to claim 1, characterized in that, The dose of the compound Gaozibo tablets is 0.0135 g / ml-0.054 g / ml.

3. Use according to claim 1, characterized in that, The application is at least one of improving the injury caused by cerebral ischemia-reperfusion or reducing the infarction area.

4. Use according to claim 1, characterized in that, The application is at least one of significantly reducing the necrosis, atrophy and necrosis of nerve cells, and effectively improving the morphology and integrity of nerve cells in the brain tissue of rats with cerebral ischemia-reperfusion injury.

5. The use according to claim 1, characterized in that, The application is significantly reducing the apoptosis cells in the brain tissue of rats with cerebral ischemia-reperfusion injury.

6. Use according to claim 1, characterized in that, The application is improving the expression of key genes in cerebral ischemia-reperfusion injury.

7. Use according to claim 6, characterized in that, The application is at least one of significantly reducing the expression of pro-inflammatory factors TNF-ɑ, IL-6, IL-1β and pro-oxidant MDA in the brain tissue of rats with cerebral ischemia-reperfusion injury, and significantly increasing SOD in the brain tissue. The application is at least one of significantly reducing the expression of pro-inflammatory factors TNF-ɑ, IL-6, IL-1β and pro-oxidant MDA in the brain tissue of rats with cerebral ischemia-reperfusion injury, and significantly increasing SOD in the brain tissue.

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