Uses of Astragalus injection in the preparation of drugs for treating cerebral hemorrhage

Abstract

This invention belongs to the field of pharmaceutical technology. Addressing the lack of reported applications of Astragalus injection in the preparation of drugs for treating cerebral hemorrhage, this invention proposes the use of Astragalus injection in the preparation of drugs for treating cerebral hemorrhage. Experimental results show that Astragalus injection can protect brain tissue neurons by inhibiting the HMGB1 / RAGE signaling pathway, inhibiting neuronal apoptosis, and reducing the expression of inflammatory factors in brain tissue, thereby achieving a protective effect against cerebral hemorrhage in rats. Therefore, Astragalus injection has the potential to be used as a drug for treating cerebral hemorrhage.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of Astragalus injection in the preparation of drugs for treating cerebral hemorrhage. Background Technology

[0002] Intracerebral hemorrhage (CH), a common acute condition of the central nervous system, has become a major challenge in global public health due to its high incidence, high risk of disability, and high mortality. CH not only directly causes mechanical damage to brain tissue but also triggers a series of secondary pathological changes, including hematoma formation, cerebral edema, and inflammatory responses. Studies have shown that neuronal apoptosis is a key factor in causing neurological dysfunction after CH. Neuronal apoptosis not only exacerbates brain damage but may also further promote brain tissue damage and functional impairment by activating various cell death pathways. Therefore, finding effective intervention strategies to reduce neuronal apoptosis after CH has become an important issue in the treatment of CH.

[0003] High-mobility group box-1 protein (HMGB1) plays a crucial role in the inflammatory response and neuronal apoptosis following intracerebral hemorrhage. HMGB1 is a non-histone DNA-binding protein that interacts with downstream signaling molecules through its receptor for advanced glycation end products (RAGE), activating multiple signaling pathways and inducing pathological processes such as cellular inflammation and apoptosis. The role of the HMGB1 / RAGE signaling pathway in cerebrovascular diseases has gradually attracted widespread attention in the academic community, and studies have shown that this pathway is closely related to neurological damage following intracerebral hemorrhage.

[0004] Astragalus, a traditional Chinese medicine, exhibits multiple biological activities, including antioxidant, anti-inflammatory, and immunomodulatory effects. Clinically, it is often used to enhance immunity and improve microcirculation. Astragalus injection, a preparation of astragalus extract, has been used in the treatment of various diseases. However, its application in improving cerebral blood flow, reducing cerebral edema, and in anti-inflammatory and antioxidant effects has not yet been reported. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the use of Astragalus injection in the preparation of drugs for treating cerebral hemorrhage.

[0006] The first aspect of this invention provides the use of Astragalus injection in the preparation of a medicament for treating cerebral hemorrhage.

[0007] Preferably, the Astragalus injection is administered clinically via intraperitoneal injection.

[0008] Preferably, the dosage of the Astragalus injection is 8 ml / kg·d.

[0009] A second aspect of the present invention provides a drug for treating cerebral hemorrhage, the drug comprising Astragalus injection.

[0010] Preferably, the drug further includes pharmaceutically acceptable excipients. A specific excipient may be polysorbate-80.

[0011] Compared with the prior art, the advantages of this invention are as follows: This invention establishes a rat model of cerebral hemorrhage and treats the rat model with Astragalus injection. Experimental results show that Astragalus injection can protect brain neurons by inhibiting the HMGB1 / RAGE signaling pathway, suppressing neuronal apoptosis, and reducing the expression of inflammatory factors in brain tissue, thereby achieving a protective effect against cerebral hemorrhage in rats. Therefore, Astragalus injection has the potential to be used as a drug for treating cerebral hemorrhage. Attached Figure Description

[0012] Figure 1 Pathological changes in brain tissue of rats in each group (HE, ×40). Figure 2 Apoptosis of nerve cells in the brain tissue of rats in each group (TUNEL, ×40). Figure 3 The images show the protein bands of HMGB1, RAGE, Bcl-2, Bcl-2, and Cleaved caspase-3 in the brain tissue of rats in each group. Detailed Implementation

[0013] The following will be described in conjunction with embodiments of the present invention. Figures 1 to 3 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0014] 1. Experimental materials 1.1 Cells PC-12 (highly differentiated) rat adrenal pheochromocytoma cell line (purchased from Zhejiang Meisen Cell Technology Co., Ltd., batch number CTCC-003-0209), abbreviated as PC12 cells.

[0015] 1.2 Animals Eight-week-old SD rats (hereinafter referred to as rats) from Guangdong Lady Biomedical Research Institute Co., Ltd. were selected as experimental subjects, with a body weight of 180 g to 220 g, production license number: SCXK (Guangdong) 2022-0064, and usage license number: SYXK (Guangdong) 2022-0296. The rats were kept at a room temperature of 20-24℃, with a relative humidity of 50% to 70%, and underwent a periodic alternation of 12 hours of light and 12 hours of darkness. They had free access to food and water and were acclimatized for 7 days before the experiment was conducted.

[0016] 1.3 Reagents and Instruments Astragalus injection (batch number 1404163, Hunan Wuzhoutong Pharmaceutical Co., Ltd.); Sodium pentobarbital (batch number T0894, Sigma-Aldrich, USA); Dexamethasone (batch number LOT20235125, Beijing Solarbio Science & Technology Co., Ltd.); Normal saline (batch number FS-P0178, Shanghai Fusheng Industrial Co., Ltd.); CCK-8 reagent kit (batch number S0918L01, Shanghai Aibisin Biotechnology Co., Ltd.); HE staining kit (batch number BP0211, Hubei Bios Biotechnology Co., Ltd.); 4% paraformaldehyde solution (catalog number P0099, Shanghai Beyotime Biotechnology Co., Ltd.); TUNEL staining kit (batch number PF00006, Proteintech); HMGB1 ELISA kit (batch number K429AD0355, Sangon Biotech (Shanghai) Co., Ltd.); RAGE ELISA kit (batch number 20160508, Nanjing Kaiji Biotechnology Co., Ltd.); Tumor necrosis factor α (Tumor Necrosis Factor α) Factor-α, TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-β) ELISA kits (lot numbers ml002859, ml064292, ml037361, Shanghai Enzyme-Link Biotechnology Co., Ltd.); interleukin-10 (IL-10); ELISA kit (lot number EMC005QT.96, Xinbosheng Biotechnology Co., Ltd.); HMGB1 antibody (lot number A00066-1, Boster Biological Technology Co., Ltd.); RAGE antibody (lot number 821904538, GeneTex, USA); B-cell lymphoma-2 (Bcl-2) antibody (catalog number 250412, Chengdu Zhengneng Biotechnology Co., Ltd.); Bcl-2-associated X protein. Bax antibody (batch number ab32124, Abcam, UK); Cleaved caspase-3 antibody (batch number 341052, Chengdu Zhengneng Biotechnology Co., Ltd.); SpectraMAX Plus384 microplate reader (Meigu Molecular Instruments Co., Ltd.); Electrophoresis apparatus (Bio-Rad, USA); RM2235 pathological slide machine (Leica, Germany); Upright optical microscope (Nikon Eclipse E100, Japan).

[0017] 2. Experimental Methods 2.1 Cell Experiments 2.1.1 Cell modeling and grouping PC12 cells were routinely cultured in a 37°C incubator containing 5% CO2 using RPMI 1640 medium (i.e., complete medium) containing 10% fetal bovine serum (FBS) and 1% penicillin antibiotics. PC12 cells in the logarithmic growth phase were selected, the complete medium was removed, and replaced with sugar-free DMEM medium. The cells were then cultured in an anaerobic incubator containing 1% O2, 94% N2, and 5% CO2 at 37°C for 4 h. Subsequently, the complete medium was replaced, and the cells were reoxygenated for 24 hours at a standard oxygen concentration (17-18%) before being analyzed and observed.

[0018] PC12 cells were divided into a control group and multiple treatment groups (administered with Astragalus injection at concentrations of 25, 50, 75, and 100 mg / mL, respectively). Both the control group and the treatment groups first underwent the aforementioned oxygen-glucose deprivation treatment, followed by reoxygenation treatment; compared to the control group, all treatment groups also received the corresponding concentration of Astragalus injection during the reoxygenation phase.

[0019] 2.1.2 CCK-8 assay for cell viability 10 mL of CCK-8 solution was added to the blank group and multiple drug-treated groups, and incubated in an incubator for 1 h to 4 h. The absorbance value was measured and recorded at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0020] 2.2 Animal Experiments 2.2.1 Modeling A rat model of cerebral hemorrhage was established using the autologous blood injection method. The specific method for establishing the rat model of cerebral hemorrhage is as follows: Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg) and then securely placed on a stereotactic brain model. The model was adjusted to ensure the anterior and posterior fontanelles were at the same level. Subsequently, the skin was incised in the central region of the head to expose the skull, and a small hole (approximately 1 mm) was drilled without damaging the dura mater. 50 µl of non-anticoagulated autologous arterial blood was drawn using a microsyringe. The microsyringe was fixed to the stereotactic brain model and inserted vertically through the pre-drilled hole to the predetermined depth. Blood was slowly and evenly injected into the rat's brain. After injection, the needle was left in place for a period of time before being slowly withdrawn, the incision sutured, and the skin disinfected. The rats exhibited abnormal motor and neurological functions, such as sluggishness, dull fur, lethargy, and decreased appetite. Most rats showed contralateral limb paralysis, unsteady gait, and some were unable to stand. A few showed dementia or drowsiness. The presence of these symptoms indicated successful model establishment.

[0021] 2.2.2 Grouping Rats were divided into a sham-operated group, a model group, an Astragalus injection group, and a positive control group. The specific methods for constructing each group of rats were as follows: Sham surgery group: Rats underwent only the skull exposure operation described in section 2.2.1 above, without any other operations. After the skull exposure operation, they were given 3 ml / (kg·d) of physiological saline intraperitoneally for 14 days. Model group: The constructed rat model of cerebral hemorrhage was administered 3 ml / (kg·d) of physiological saline intraperitoneally daily for 14 days; Astragalus injection group: The constructed rat model of cerebral hemorrhage was given 8 ml / (kg·d) of Astragalus injection (1 ml of Astragalus injection = 2 g of Astragalus crude drug) via intraperitoneal injection for 14 days; Positive control group: The constructed rat model of cerebral hemorrhage was administered dexamethasone 0.75 mg / (kg·d) via intraperitoneal injection for 14 days.

[0022] 2.2.3 Calculate brain tissue water content and brain index After decapitation, the brains of rats in each group were removed and weighed immediately, recorded as the initial weight W1. The brains were then dried in an 80℃ oven until constant weight. After being removed and allowed to cool to room temperature, they were weighed again, recorded as W2. The water content and brain index of the brain tissue were obtained from W1 and W2. The formulas for calculating the water content and brain index of brain tissue are: The water content of brain tissue = (W1 - W2) / W1 × 100%; Brain index = W1 / body weight × 100%.

[0023] 2.2.4. Longa 5-point scoring system for assessing rat neurological function 0 points (normal): The rats behave normally, have no neurological deficits, and can walk freely; 1 point (mild neurological deficit): The rat showed mild motor impairment, such as mild paralysis of one side of the limbs. It could walk but its gait was somewhat unsteady. 2 points (moderate neurological deficit): The rat has partial weakness or paralysis on one side of its limbs and cannot walk, but can move slightly with the help of other limbs; 3 points (severe neurological deficit): The rats showed obvious hemiplegia, were completely unable to walk, and had limited mobility. They may have lost their balance or be unable to stand. 4 points (extremely severe neurological deficit): The rat completely loses its ability to move, is unable to stand or move, and is extremely weak or in a state of deep sleep.

[0024] 2.2.5. Pathological observation of rat brain tissue was performed using HE staining technique. Brain tissue samples were extracted and then fixed in 4% paraformaldehyde solution for 48 hours. Following dehydration and clearing, the brain tissue was embedded in paraffin and cut into 4-5 micrometer thick paraffin sections using an RM2235 pathological slide machine. The sections were baked in a 60 °C oven for 3 hours, dewaxed with xylene, immersed in graded ethanol, rinsed three times, primary stained with hematoxylin for 5 minutes, counterstained with eosin for 4 minutes, and mounted with neutral resin. The pathological changes in the brain tissue of each group of rats were then carefully observed under an upright optical microscope.

[0025] 2.2.6. TUNEL staining to observe apoptosis of nerve cells in rat brain tissue Paraffin sections of 4-5 μm brain tissue were prepared and treated with proteinase K to remove proteins and expose DNA break sites. Enzymatic reactions were performed using TdT enzyme and labeled dUTP from the TUNEL staining kit to label the 3'-OH ends of DNA breaks. Nucleic acids were stained with DAPI, and fluorescently labeled apoptotic cells in the sections were observed. Detection and analysis were performed under a fluorescence microscope to assess the apoptosis status of nerve cells.

[0026] 2.2.7. Serum levels of HMGB1, RAGE, TNF-α, IL-6, IL-10, and IL-β in rats were detected by ELISA. Before sacrificing the rats, 2 ml of blood was collected from their abdominal aorta and centrifuged at 3000 rpm for 15 minutes to separate the serum supernatant. Subsequently, the levels of cytokines such as HMGB1, RAGE, TNF-α, IL-6, IL-10, and IL-1β in the serum were detected using an ELISA kit.

[0027] 2.2.8. Western blotting method was used to detect the expression of HMGB1, RAGE, and apoptosis-related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2-related X protein (Bax), and cleaved caspase-3 in rat brain tissue. Total protein was extracted from rat brain tissue. Specifically, the rat brain tissue was lysed using RIPA protein lysis buffer, and the concentration of the obtained protein was determined by the BCA method. Proteins were separated by gel electrophoresis and transferred to appropriate carriers. After transfer, the carriers were blocked with 5% skim milk powder. Rabbit primary antibodies (1:1000) targeting HMGB1, RAGE, Bcl-2, Bax, and Cleaved caspase-3 were added and incubated overnight at 4°C. The next day, goat anti-rabbit HRP secondary antibody (1:3000) was added and incubated at room temperature for 1 h. Protein bands were observed using a gel imaging system, and the grayscale values ​​of the bands were quantitatively evaluated using ImageJ software.

[0028] 2.3 Statistical Analysis Statistical analysis was performed on the obtained data using SPSS version 26.0 software. For continuous variable data, this invention uses the mean and standard deviation (...) Data were described in the form of ( ), and differences between groups were analyzed using one-way ANOVA and LSD-t test. Data that did not conform to a normal distribution were characterized using the median (M) and interquartile ranges (P25, P75), and subjected to the Kruskal-Wallis nonparametric test. The significance level was set at P=0.05.

[0029] 3. Experimental Results 3.1 Effect of Astragalus injection on OGD / R-induced PC12 cell viability The CCK-8 test results are shown in Table 1 below.

[0030] Table 1 Comparison of nerve cell activity in rats of different groups ( (n=3)

[0031] The results in Table 1 show that, compared with the control group, the activity of PC12 cells in the drug-treated group was significantly increased ( P <0.05); In the treatment group, PC12 cell activity increased significantly with increasing dose ( P <0.05), the results showed that within a specific concentration range, the concentration of Astragalus injection had a good positive correlation with its protective effect on cells.

[0032] 3.2 Comparison of brain tissue water content and brain index among different groups of rats The results of brain tissue water content and brain index of rats in each group are shown in Table 2.

[0033] Table 2 Comparison of brain tissue water content and brain index in rats of different groups ( (n=10)

[0034] Note: a: Compared with the sham surgery group P <0.05; b: Compared with the model group, P <0.05, the same applies below; The results in Table 2 show that, compared with the sham-operated group, the brain tissue water content and brain index of the model group rats were both increased ( P <0.05); compared with the model group, the water content and brain index of rat brain tissue in the Astragalus injection group and the positive control group were decreased ( P <0.05).

[0035] 3.3 Comparison of neurological function scores among different groups of rats The comparison results of the neurological function scores of rats in each group are shown in Table 3.

[0036] Table 3 Comparison of neurological function scores among different groups of rats ( (n=10)

[0037] The results in Table 3 show that, compared with the sham-operated group, the neurological function scores of rats in the model group were higher ( P <0.05); compared with the model group, the neurological function scores of rats in the Astragalus injection group and the positive control group were reduced ( P <0.05).

[0038] 3.4 Pathological changes in the brain tissue of rats in each group pass Figure 1 It can be seen that the brain tissue of the sham-operated group rats was intact, with regular neuronal distribution, normal cells, no nuclear pyknosis or fragmentation, and no edema or necrosis. The brain tissue of the model group rats showed obvious lesions, including edema, structural disorder, erythrocyte aggregation, inflammatory cell infiltration, and swelling and degeneration of nerve cells. In severe cases, cell apoptosis and necrosis occurred. The nerve cell swelling, erythrocyte aggregation, and inflammatory cell infiltration of the Astragalus injection group and the positive control group rats were improved, and the degree of lesions was lower than that of the model group.

[0039] 3.5 Apoptosis of nerve cells in the brain tissue of rats in each group The comparison results of apoptosis rates of nerve cells in the brain tissue of rats in each group are shown in Table 4.

[0040] Table 4 Comparison of apoptosis rates of nerve cells in rat brain tissue among different groups ( (n=10)

[0041] The results in Table 4 show that, compared with the sham-operated group, the apoptosis rate of nerve cells in the brain tissue of rats in the model group was increased ( P<0.05); compared with the model group, the apoptosis rate of nerve cells in the brain tissue of rats in the Astragalus injection group and the positive control group decreased ( P <0.05).

[0042] pass Figure 2 It can be seen that the sham-operated group had more blue-stained cells and fewer apoptotic cells in the field of view, and the overall condition was better. The model group had more dark-colored apoptotic cells in the field of view, indicating that the apoptosis of nerve cells was significantly increased and the degree of cell damage was higher. The Astragalus injection group and the positive control group had fewer apoptotic cells in the field of view than the model group, and the degree of cell damage was reduced. This shows that Astragalus injection not only plays a positive role in improving brain tissue structure, but also can reduce the apoptosis rate of nerve cells by regulating the balance between cell survival and death, thereby protecting brain function.

[0043] The above results indicate that the brain tissue water content and brain index of rats in the Astragalus injection group and the positive control group were lower than those in the model group, and the neurological function score and neuronal apoptosis rate were also significantly reduced. This suggests that Astragalus injection can effectively improve the pathological damage of brain tissue in rats with cerebral hemorrhage and alleviate the symptoms of neurological dysfunction.

[0044] 3.6 Comparison of serum HMGB1, RAGE, TNF-α, IL-6, IL-10, and IL-β levels in rats from different groups The results in Table 5 show that, compared with the sham-operated group, the serum levels of HMGB1, RAGE, TNF-α, IL-6, and IL-β were increased, while the IL-10 level was decreased in the model group rats. P <0.05), suggesting that the HMGB1 / RAGE signaling pathway plays an important role in the inflammatory response and neuronal apoptosis after cerebral hemorrhage; compared with the model group, the serum levels of HMGB1, RAGE, TNF-α, IL-6, and IL-β were decreased in the Astragalus injection group and the positive control group, while the IL-10 level was increased ( P <0.05), indicating that Astragalus injection can inhibit the activation of the HMGB1 / RAGE signaling pathway, regulate the expression of inflammatory factors in rats with cerebral hemorrhage, reduce the intensity of inflammatory response, and thus alleviate the degree of brain tissue damage.

[0045] Table 5. Comparison of serum HMGB1, RAGE, TNF-α, IL-6, IL-10, and IL-β levels in rats from different groups ( (n=10)

[0046] 3.7 Expression of HMGB1, RAGE, Bcl-2, Bax, and Cleaved caspase-3 proteins in rat brain tissue of each group Through Table 6 and Figure 3The results showed that, compared with the sham-operated group, the expression of HMGB1, RAGE, Bax, and Cleaved caspase-3 proteins in the brain tissue of rats in the model group was increased, while the expression of Bcl-2 protein was decreased. P <0.05), suggesting activation of the HMGB1 / RAGE pathway or imbalance of apoptotic molecules, exacerbating neuronal apoptosis; compared with the model group, the expression of HMGB1, RAGE, Bax, and Cleaved caspase-3 proteins in the brain tissue of rats in the Astragalus injection group and the positive control group decreased ( P <0.05), Bcl-2 protein expression increased ( P <0.05), suggesting that Astragalus injection can inhibit the apoptosis process of nerve cells by regulating the expression of key proteins in this signaling pathway. This mechanism provides a molecular explanation for the neuroprotective effect of Astragalus after cerebral hemorrhage.

[0047] Table 6. Expression of HMGB1, RAGE, Bcl-2, and Cleaved caspase-3 proteins in rat brain tissue of each group ( (n=3)

[0048] In summary, this invention demonstrates that Astragalus injection has a significant neuroprotective effect on rats with cerebral hemorrhage. Specifically, it may achieve the treatment of cerebral hemorrhage by inhibiting the HMGB1 / RAGE signaling pathway and regulating the expression of inflammatory factors and apoptotic proteins. These research results provide new insights and theoretical support for clinical treatment strategies for cerebral hemorrhage.

[0049] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. Use of Astragalus injection in the preparation of drugs for treating cerebral hemorrhage.

2. The use according to claim 1, characterized in that, The Astragalus injection is administered clinically via intraperitoneal injection.

3. The use according to claim 2, characterized in that, The dosage of the Astragalus injection is 8 ml / kg·d.

4. A drug for treating cerebral hemorrhage, characterized in that, The drug includes Astragalus injection.

5. The drug for treating cerebral hemorrhage according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images