Application of Siegesbeckia orientalis and Astragalus membranaceus composition in the preparation of drugs to improve cognitive impairment after heart failure

Abstract

This invention discloses the application of the Siegesbeckia orientalis composition in the preparation of drugs to improve cognitive impairment after heart failure (CHF). The composition consists of Astragalus membranaceus extract, Siegesbeckia orientalis extract, and Periploca sepium extract. Experiments have shown that the Siegesbeckia orientalis composition can significantly improve spatial memory and working memory in CHF rats, and improve cognitive function; it can significantly inhibit the release of tumor necrosis factor α and interleukin 1β in the hippocampus of CHF rats, and increase the release of interleukin 10 and brain-derived neurotrophic factor; it can significantly reduce the loss and damage of Nissl bodies in the brain tissue of CHF rats; and it can significantly improve the ultrastructure of neurons in the hippocampus of CHF rats, regulate synaptic plasticity, and promote the growth of dendrites and dendritic spines.

Description

Technical Field

[0001] This invention relates to novel uses of Siegesbeckia orientalis compositions, particularly their use in the preparation of drugs for improving cognitive impairment following heart failure. Background Technology

[0002] Post-heart failure cognitive impairment refers to the symptoms and signs of memory impairment, attention difficulties, and attention deficit following chronic heart failure (CHF). CHF is a group of clinical syndromes caused by various structural or functional cardiac diseases leading to impaired ventricular filling and / or ejection capacity. More than 40%-60% of CHF patients exhibit cognitive impairment, mainly manifested as learning and working memory deficits, delayed recall, depression, and anxiety. These cognitive impairments are highly correlated with the hippocampus, amygdala, and prefrontal cortex. There are currently 4.5 million CHF patients in my country, with approximately 500,000 new cases each year. With the increasing aging of the population, the prevalence of CHF is showing a significant upward trend. Due to lower cognitive reserve in the elderly, CHF patients are more prone to developing cognitive impairment. These cognitive impairments further deteriorate the quality of life and self-care of CHF patients. Compared to CHF patients without comorbid cognitive impairment, CHF patients with cognitive impairment have significantly higher readmission and mortality rates. In recent years, modern medicine has made significant progress in the prevention and treatment of CHF (congenital heart disease). However, treatment methods and interventions for post-CHF cognitive impairment are insufficient, and the overall prognosis remains poor. Therefore, there is an urgent need to explore new therapies for preventing and treating post-CHF cognitive impairment.

[0003] Traditional Chinese medicine (TCM) possesses the characteristics of multi-pathway, multi-component, multi-target, and good safety profile, demonstrating significant efficacy in the prevention and treatment of chronic heart failure (CHF) and cognitive impairment. Clinical practice has proven that TCM has certain advantages in improving clinical symptoms, enhancing quality of life, improving cardiac function, reversing ventricular remodeling, and slowing the progression of CHF, thus exhibiting clear therapeutic value for CHF. Based on the theory of "treating different diseases with the same method," TCM can significantly improve cognitive functions such as memory, executive function, and attention in CHF patients while simultaneously improving CHF. TCM considers cognitive impairment to fall under the categories of "forgetfulness," "morbid forgetfulness," and "dullness" in traditional Chinese medicine, representing a syndrome of deficiency in the root and excess in the branch. The root deficiency is primarily due to qi and blood deficiency and malnourishment of the marrow, while the branch excess is often caused by wind, phlegm, and blood stasis. The syndrome elements tend to be a mixture of deficiency and excess. Among these, qi deficiency and phlegm turbidity are closely related to the progression of cognitive impairment after heart failure. The main treatment principles are tonifying qi and blood, replenishing kidney essence, resolving phlegm, and unblocking the meridians.

[0004] Currently, there are no reports of using the combination of Siegesbeckia or Astragalus membranaceus to improve cognitive impairment after heart failure. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of the Siegesbeckia orientalis composition in the preparation of drugs to improve cognitive impairment after heart failure.

[0006] The technical solution of this invention is summarized as follows:

[0007] The use of the Siegesbeckia orientalis composition in the preparation of a drug to improve cognitive impairment after heart failure, wherein the Siegesbeckia orientalis composition is composed of Astragalus membranaceus extract, Siegesbeckia orientalis extract and Periploca sepium extract.

[0008] Preferably, the mass ratio of Astragalus membranaceus extract, Siegesbeckia orientalis extract and Periploca sepium extract is (17-19):(2-4):1.

[0009] Advantages of this invention:

[0010] (1) Experiments have shown that the combination of Siegesbeckia orientalis and Astragalus membranaceus can significantly improve spatial memory and working memory in CHF rats and improve cognitive function. (2) The combination of Siegesbeckia orientalis and Astragalus membranaceus can significantly inhibit the release of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in the hippocampus of CHF rats, and increase the release of interleukin 10 (IL-10) and brain-derived neurotrophic factor (BDNF).

[0011] (3) The combination of Siegesbeckia orientalis and Astragalus membranaceus can significantly reduce the loss and damage of Nissl bodies in the brain tissue of CHF rats.

[0012] (4) The combination of Siegesbeckia orientalis and Astragalus membranaceus can significantly improve the ultrastructure of neurons in the hippocampus of CHF rats, regulate synaptic plasticity, and promote the growth of dendrites and dendritic spines. Attached Figure Description

[0013] Figure 1 The effects of the combination of Siegesbeckia orientalis and Astragalus membranaceus on cognitive function in CHF rats.

[0014] Figure 2 Effects of Siegesbeckia orientalis and Astragalus membranaceus combination on TNF-α, IL-1β, IL-10 and BDNF in the hippocampus of CHF rats.

[0015] Figure 3 The effects of the combination of Siegesbeckia orientalis and Astragalus membranaceus on the morphology and number of Nissl bodies in the hippocampus of CHF rats.

[0016] Figure 4 Effects of the combination of Siegesbeckia orientalis and Astragalus membranaceus on the ultrastructure of neurons in the hippocampus of CHF rats.

[0017] Figure 5The effects of the combination of Siegesbeckia orientalis and Astragalus membranaceus on the synaptic structure and function of the hippocampus in CHF rats.

[0018] Figure 6 Effects of Siegesbeckia orientalis and Astragalus membranaceus combination on dendritic growth in the hippocampus of CHF rats.

[0019] Figure 7 Effects of Siegesbeckia orientalis and Astragalus membranaceus combination on dendritic spine growth in the hippocampus of CHF rats. Detailed Implementation

[0020] The Astragalus membranaceus extract, Siegesbeckia orientalis extract, and Periploca sepium extract of the various embodiments of the present invention were purchased from Beijing Kangrentang Pharmaceutical Co., Ltd. (batch number: 22025921), Beijing Kangrentang Pharmaceutical Co., Ltd. (batch number: 22010811), and Beijing Kangrentang Pharmaceutical Co., Ltd. (batch number: 21004811). The sources of the extracts in the formulations of the present invention are disclosed for the purpose of better illustrating the present invention, but the sources of the extracts of the present invention are not limited.

[0021] The combination of Siegesbeckia or Astragalus membranaceus, abbreviated as XJQ, can also be called Siegesbeckia or Astragalus membranaceus formula.

[0022] Preparation of the Siegesbeckia orientalis-astragalus combination: The astragalus extract, sisal extract and citronella bark extract are mixed together to obtain the product.

[0023] Example 1

[0024] The Siegesbeckia orientalis composition consists of 18.06 g of Astragalus membranaceus extract, 3.17 g of Siegesbeckia orientalis extract and 1 g of Periploca sepium extract.

[0025] Example 2

[0026] The Siegesbeckia orientalis composition consists of 17 grams of Astragalus membranaceus extract, 4 grams of Siegesbeckia orientalis extract, and 1 gram of Periploca sepium extract.

[0027] Example 3

[0028] The Siegesbeckia orientalis composition consists of 19 grams of Astragalus membranaceus extract, 2 grams of Siegesbeckia orientalis extract, and 1 gram of Periploca sepium extract by weight.

[0029] Example 4

[0030] The method for establishing a rat CHF model includes the following steps:

[0031] Male SD rats, 8 weeks old, 220±10g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0032] (1) Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (60 mg / kg);

[0033] (2) Use a 16G intravenous catheter to insert a tracheal tube and connect it to a small animal ventilator;

[0034] (3) Open the heart in the 3rd-4th intercostal space of the rat and ligate the left anterior descending coronary artery with a 3 / 8 curved needle 2 mm below the intersection of the left atrial appendage and the pulmonary artery conus. Ischemia in the myocardial region is confirmed by visual observation (whitening of color) and electrocardiogram monitoring (ST segment elevation and QRS widening).

[0035] (4) Suture the intercostal muscles, muscles and skin layer by layer from the inside out to close the thoracic cavity.

[0036] Inclusion criteria for CHF rats: 24 hours after surgery, the rats had 6-8 pathological Q waves on a 12-lead electrocardiogram.

[0037] Example 5

[0038] Grouping and administration: The drugs were prepared in Example 1 and included the following steps:

[0039] (1) Fifty male SD rats were selected and randomly divided into 5 groups of 10 rats each, according to the number of pathological Q waves on the electrocardiogram 24 hours after surgery:

[0040] ①Sham surgery group;

[0041] ② CHF group (CHF);

[0042] ③ The low-dose combination of Siegesbeckia orientalis and Astragalus membranaceus (0.778 g / kg) + CHF group (XJQL+CHF);

[0043] ④ The combination of Siegesbeckia orientalis and Astragalus membranaceus at a medium dose (1.556 g / kg) + CHF group (XJQM+CHF);

[0044] ⑤ High-dose combination of Siegesbeckia orientalis and Astragalus membranaceus (3.112 g / kg) + CHF group (XJQH+CHF);

[0045] The Sham group underwent the same surgical procedure, but the sutures were threaded through without ligation.

[0046] The group receiving the Siegesbeckia orientalis and Astragalus membranaceus combination was given the drug via gavage starting 24 hours after surgery, once daily for 6 consecutive weeks; the Sham group and CHF group were given an equal volume of normal saline via gavage for 6 consecutive weeks.

[0047] (2) After 6 weeks, the cognitive function, inflammatory factors, Nissl bodies, neuronal and synaptic ultrastructure, dendrites, and dendritic spine growth of rats in each group were examined.

[0048] Example 6

[0049] Effects of Siegesbeckia orientalis and Astragalus membranaceus combination on cognitive function in CHF rats.

[0050] Experimental materials

[0051] Instruments: The Morris water maze experimental setup was manufactured by Shanghai Yishu Co., Ltd. (XR-XM101). The setup mainly includes a black circular water tank (diameter 120cm, height 40cm); a small transparent circular platform that is easy to move and disassemble (platform diameter 8cm of the water tank); an automatic tracking and acquisition camera; a monitor; and a computer equipped with an analysis and processing system.

[0052] Experimental methods

[0053] Test content: This part of the experiment is mainly divided into two parts: positioning and navigation experiment and space exploration experiment.

[0054] Test Environment and Equipment Requirements: The Morris water maze test requires a noise-free environment, free from direct light, and the lighting level in the test room should remain relatively constant throughout the test (use blackout curtains to block stray light and avoid interference with image acquisition). Before the test, fill the pool with tap water, maintaining a water temperature of 22℃-24℃. The water level should be kept 1cm above the platform so that the rats cannot see the underwater platform. Additionally, pour blue-black ink into the pool and stir thoroughly to make the water surface black during the test (the pool water color must contrast sharply with the color of the white SD rats to ensure the camera functions correctly and accurately for tracking and acquisition).

[0055] Specific testing steps: ① Adaptation training: First, place the rat on a platform hidden underwater for 5 seconds. Then, place the rat into the pool facing the pool wall from any quadrant. If the rat does not find the platform within 60 seconds, place it on the platform again for 5 seconds to reinforce its memory. No further training is performed after this. ② Positional navigation experiment: The platform location is fixed. Each day, rats are randomly placed into the pool facing the pool wall from the remaining three quadrants. The time it takes to find the hidden platform (escape latency), total swimming distance, swimming speed, and other parameters are recorded. Recordings are repeated for 5 consecutive days, with each quadrant being tested once per day. ③ Spatial exploration experiment: After the positioning navigation experiment, the platform is removed. One of the remaining quadrants is randomly selected as the entry point. The rat's swimming trajectory is recorded over a certain period, mainly recording the number of times the rat crosses the platform and its swimming speed.

[0056] Experimental results

[0057] The orientation and heading parameters of each group of rats are shown in Table 1.

[0058] Table 1. Plateau latency, total swimming distance, and average speed of rats in each experimental group.

[0059]

[0060] Note: *P<0.05, ** P<0.01 vs. Sham group # P<0.05, ## P<0.01 vs. CHF group, n=10.

[0061] As shown in Table 1, compared with the Sham group, the plateau latency of rats in the CHF group was ( Figure 1 A) Relative lengthening, total swimming distance and average speed ( Figure 1 B and 1C) showed no significant changes; compared with the CHF group, the Siegesbeckia orientalis composition of Example 1 of the present invention could shorten the plateau latency period to varying degrees at different doses.

[0062] The parameters of the spatial exploration experiment for each group of rats are shown in Table 2.

[0063] Table 2. Number of times rats crossed the platform, percentage of time spent in the target quadrant, and percentage of distance traveled in the target quadrant for each experimental group.

[0064]

[0065] Note: * P<0.05, ** P<0.01 vs. Sham group # P<0.05, ## P<0.01 vs. CHF group, n=10.

[0066] As shown in Table 2, compared with the Sham group, the CHF group rats had fewer platform crossings ( Figure 1 D) Target quadrant time percentage ( Figure 1 E) and the percentage of the distance to the target quadrant ( Figure 1 F) significantly decreased (P<0.05); compared with the CHF group, different doses of the Siegesbeckia orientalis composition of Example 1 of the present invention could increase the number of times crossing the platform, the percentage of time in the target quadrant, and the percentage of distance traveled in the target quadrant to varying degrees. Among them, the medium and high dose groups of the Siegesbeckia orientalis composition showed statistically different effects on the number of times crossing the platform, the percentage of time in the target quadrant, and the percentage of distance traveled in the target quadrant compared with CHF (P<0.05); this result indicates that the Siegesbeckia orientalis composition can significantly improve the cognitive function of CHF rats.

[0067] Example 7

[0068] Effects of Siegesbeckia orientalis and Astragalus membranaceus combination on TNF-α, IL-1β, IL-10 and BDNF in the hippocampus of CHF rats.

[0069] Experimental materials

[0070] (1) Reagents: Rat tumor necrosis factor α (TNF-α) ELISA kit (DG20065D), rat interleukin-1β

[0071] (IL-1β) ELISA kit (DG20049D), rat interleukin-10 (IL-10) ELISA kit (DG20120D), rat brain-derived neurotrophic factor (BDNF) ELISA kit (DG20046D).

[0072] (2) Instruments: 37℃ constant temperature and humidity chamber, microplate reader (Rayto RT-6100, Rayto, USA).

[0073] Experimental methods

[0074] (1) Tissue sample collection and processing

[0075] ①Six weeks after the operation, the rats in each group were decapitated and their brains were removed under deep anesthesia;

[0076] ② Carefully separate the rat brain hippocampus, accurately weigh it, and place it in a homogenizer. Add 9 times the weight-to-volume ratio of physiological saline and grind it thoroughly to make a tissue homogenate. Centrifuge at 3000 rpm for 10 minutes and take the supernatant, which is 10% brain tissue homogenate.

[0077] ② Take 0.1 mL of 10% tissue homogenate, add 0.1 mL of phosphate buffer and mix well. Centrifuge at 3000 rpm for 10 minutes and take the supernatant for testing.

[0078] (2) ELISA operation steps

[0079] ① The reagent kit should be removed from the refrigerated environment and allowed to equilibrate at room temperature;

[0080] ② Remove the required microplate strips from the aluminum foil bag after equilibration at room temperature for 20 minutes;

[0081] ③ Set up standard wells and sample wells, and add 50 μL of standard at different concentrations to each standard well;

[0082] ④ Add 50 μL of the sample to be tested to the sample wells, and do not add any to the blank wells;

[0083] ⑤ Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C for 60 minutes.

[0084] ⑥ Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 5 times.

[0085] ⑦ Add 50 μL each of substrate chromogenic agent A and B to each well and incubate at 37°C in the dark for 15 minutes;

[0086] ⑧ Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0087] (3) Calculation of experimental results

[0088] Plot a standard curve on graph paper or using relevant software, with the OD value of the measured standard on the x-axis and the concentration value of the standard on the y-axis, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the concentration of the sample.

[0089] Experimental results

[0090] The levels of TNF-α, ILβ, IL10 and BDNF in the hippocampus of rats in each group are shown in Table 2.

[0091] Table 3. Content of TNF-α, IL-1β, IL-10 and BDNF in the hippocampus of rats in each experimental group.

[0092]

[0093] Note: * P<0.05, ** P<0.01 vs. Sham group # P<0.05, ## P<0.01 vs. CHF group, n=6.

[0094] As shown in Table 3, compared with the Sham group, the CHF group had lower levels of TNF-α ( Figure 2 A) and IL-1β Figure 2 The content of B) was significantly increased (P < 0.05), and IL-10 was slightly increased ( Figure 2 C), BDNF Figure 2 The content of D) was significantly reduced (P<0.05); compared with the CHF group, different doses of the Siegesbeckia orientalis composition of Example 1 of the present invention could reduce the content of TNF-α and IL-1β in the hippocampus of CHF rats to varying degrees (P<0.05), while increasing the content of IL-10 and BDNF (P<0.05); the results indicate that the Siegesbeckia orientalis composition can significantly alleviate neuroinflammation in the hippocampus of CHF-induced cognitive impairment rats and increase the expression of neurotrophic factors.

[0095] Example 8

[0096] Effects of the combination of Siegesbeckia orientalis and Astragalus membranaceus on Nissl bodies in the hippocampus of rats with cognitive impairment after CHF.

[0097] Experimental materials

[0098] (1) Reagents: 4% paraformaldehyde, ethanol, xylene, and toluidine blue staining solution.

[0099] (2) Instruments: Leica ASP300S fully automatic dehydrator, paraffin embedding machine, rotary paraffin slicer, optical microscope.

[0100] Experimental methods

[0101] Six weeks post-surgery, rat hearts were harvested via thoracotomy and fixed with 4% paraformaldehyde for 48 hours. The hearts were then dehydrated in an automated dehydrator using 50%-70%-80%-95%-100% ethanol in stages, cleared with xylene, and embedded in paraffin to prepare paraffin blocks. Finally, continuous paraffin sections of 5 μm thickness were prepared using a rotary sectioner. Nissl staining procedures are as follows:

[0102] ①Preparation: Place the Nissl stained sections in a 60℃ baking oven and bake for 60 minutes.

[0103] ② Dewaxing: Place the prepared paraffin sections in xylene for dewaxing for 15 minutes; then begin gradient hydration from high to low concentration: immerse in each solution tank for 5 minutes in the order of anhydrous ethanol, 95% ethanol, 90% ethanol, 70% ethanol, 60% ethanol, and 50% ethanol. After the above steps are completed, rinse with distilled water for 5 minutes.

[0104] ③ Staining: After heating a 1% toluidine blue aqueous solution to about 50°C, place the slides into the solution, put them in an incubator, adjust the temperature to 50-60°C, and let them sit for 30-60 minutes for staining.

[0105] ④ Dehydration: Dehydrate step by step according to the gradient from low concentration to high concentration, soaking in 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol and anhydrous ethanol for 5 minutes respectively;

[0106] ⑤ Clearing: Soak the dehydrated sections in xylene twice, 10 minutes each time, and then mount them with neutral resin.

[0107] ⑥ Take photos: Observe the condition and quantity of Nissl bodies in the hippocampus under a microscope, and take photos at an appropriate magnification to record the data.

[0108] ⑦ Use ImageJ software to perform counting analysis on the CA1, CA3, and DG regions of the hippocampus.

[0109] Experimental results

[0110] The number of Nissl bodies in the hippocampus of each group of rats is shown in Table 4.

[0111] Table 4. Number of Nissl bodies in the hippocampus of rats in each experimental group

[0112]

[0113] Note: * P<0.05, ** P<0.01 vs. Sham group # P<0.05, ## P<0.01 vs. CHF group, n=4.

[0114] like Figure 3 As shown in Figure A, the Nissl bodies in the hippocampus of rats in the Sham group had normal morphology and were densely arranged. Compared with the Sham group, rats in the CHF group had more vacuoles, edema, and damage to Nissl bodies in the CA1, CA3, and DG regions of the hippocampus. (See Table 4 and...) Figure 3 The results showed that, compared with the Sham group, the number of Nissl bodies in the hippocampus of CHF group rats was reduced (P < 0.05). Different doses of the Siegesbeckia orientalis and Astragalus membranaceus combination in Example 1 of this invention could alleviate the damage and loss of Nissl bodies induced by CHF to varying degrees (P < 0.05).

[0115] Example 10

[0116] Effects of the combination of Siegesbeckia orientalis and Astragalus membranaceus on the ultrastructure and synaptic morphology of neurons in the hippocampus of CHF rats.

[0117] Experimental materials

[0118] (1) Reagents: physiological saline, glutaraldehyde fixative, 1% osmium tetroxide, 1% uranium acetate, acetone.

[0119] (2) Instruments: Leica microtome, diamond scalpel, transmission electron microscope.

[0120] Experimental methods

[0121] ① Brain tissue sampling method: Under deep anesthesia, the skull of the rat is opened to expose the brain. A few drops of fixative are dripped on it. At the same time, several cuts are made on the brain with a sharp blade, forming a "well" shape, about 2 mm deep. After the brain tissue hardens slightly, the brain is removed and the hippocampus is separated. The tissue is cut into several strips of 1*1*2 mm.

[0122] ② Transmission electron microscopy sample preparation procedure: Rinsing: Rinse with 0.1M PBS and shake thoroughly for 15 minutes × 3 times; Post-fixation: Shake with 1% osmium tetroxide for 5 minutes, then incubate at 37℃ for 1 hour; Block staining: Stain tissue blocks with 1% uranium acetate, shake continuously for 5 minutes, then let stand for 2 hours; Gradient dehydration: Dehydrate once with 50%, 70%, 80%, and 90% acetone gradients, 15 minutes each, then twice with 100% acetone, 10 minutes each; Infiltration: First, acetone: embedding solution = 1:1, incubate at 37℃ for 2 hours, then acetone: embedding solution = 1:4; incubate overnight at 37℃, and finally heat the embedding solution in a 45℃ oven for 2 hours; Embedding polymerization: Incubate at 45℃ for 3 hours, then incubate at 65℃ for 48 hours.

[0123] ③ Slide the tissue into thin sections, stain with lead citrate to observe the tissue fixation effect and neuronal distribution, and select appropriate tissue blocks and sites;

[0124] ④ Copper mesh preparation: Take a petri dish and place two layers of filter paper at the bottom. Place the newly purchased copper mesh on the filter paper and soak it in acetone for 10 minutes, shaking it gently a few times. After discarding the acetone, rinse it once with distilled water, then discard the distilled water and soak it in anhydrous ethanol for 1 minute. After shaking it gently a few times, discard the anhydrous ethanol. Let the copper mesh air dry naturally, then transfer it to a 45℃ oven to continue drying for 1 hour.

[0125] ⑤ Ultrathin sections were prepared for transmission electron microscopy to observe neuronal morphology and synaptic ultrastructure.

[0126] Experimental results

[0127] Effects of Siegesbeckia orientalis and Astragalus membranaceus combination on synaptic structural parameters in the hippocampus of CHF rats.

[0128] Table 5 Synaptic structural parameters of the hippocampus in rats of each experimental group

[0129]

[0130] Note: * P<0.05, ** P<0.01 vs. Sham group # P<0.05, ## P<0.01 vs. CHF group, n=3.

[0131] like Figure 4As shown, neurons in the Sham group exhibited slight edema, intact membranes, abundant intracellular matrix, and slightly swollen organelles. The nuclei (N) were oval, with clear, intact nuclear membranes, normal perinuclear spaces, and homogeneous chromatin. Mitochondria (M) showed mild swelling, intact membranes, present cristae, and locally faded matrix. No abnormalities were observed in the rough endoplasmic reticulum (RER) or Golgi apparatus (GO). Neurons in the CHF group showed severe edema, localized membrane damage, significant organelle swelling, and localized matrix dissolution around the cells; mitochondria showed severe swelling, matrix dissolution, disappearance of cristae, and vacuolation. The Golgi apparatus was hypertrophic, and the rough endoplasmic reticulum was significantly dilated and degranulated. Compared to the CHF group, the high-dose group of the Siegesbeckia orientalis-based combination showed significantly reduced neuronal edema and organelle swelling, intact membranes, improved mitochondrial swelling, localized membrane bulging, and partial recovery of cristae. The results indicate that the Siegesbeckia orientalis-based combination of Example 1 of this invention significantly alleviated CHF-induced neuronal damage in the hippocampus.

[0132] Figure 5 A shows the synaptic characteristic diagrams for each group. As shown in Table 5, compared to the Sham group, the CHF group had a higher synaptic density (…). Figure 5 B) Length of active region ( Figure 5 C) and the thickness of postsynaptic dense material ( Figure 5 D) significantly decreased, synaptic cleft ( Figure 5 F) significantly increased (P < 0.05); compared with the CHF group, the Siegesbeckia orientalis and Astragalus membranaceus composition (high dose) of Example 1 of this invention can increase synaptic density, active region length and postsynaptic dense material thickness (P < 0.05), and decrease synaptic cleft (P < 0.05); synaptic curvature of each group ( Figure 5 E) No significant changes. This result indicates that the combination of Siegesbeckia orientalis and Astragalus membranaceus can significantly reduce the damage to synaptic structure and function in the hippocampus of rats with cognitive impairment after CHF.

[0133] Example 11

[0134] Effects of Siegesbeckia orientalis and Astragalus membranaceus combination on the growth of dendrites and dendritic spines in the hippocampus of CHF rats.

[0135] Experimental materials

[0136] (1) Reagents: FD Rapid GolgiStain™ Kit (solutions A, B, C, D, E), double-distilled water, ethanol, xylene, resin mounting medium.

[0137] (2) Instruments: Leica ASP300S fully automatic dehydrator, paraffin embedding machine, rotary paraffin slicer, optical microscope.

[0138] Experimental methods

[0139] ① Tissue preparation: Rats were euthanized under deep anesthesia, and brain tissue was quickly removed from the skull, taking care to avoid damaging or compressing the tissue during the procedure; the blood on the surface of the tissue was quickly rinsed off with double-distilled water.

[0140] ② Immerse the brain tissue in an infiltration solution consisting of equal volumes of solutions A and B, and store in the dark at room temperature for two weeks. Replace the infiltration solution after 6 hours or the next day; transfer the brain tissue to solution C and store in the dark at room temperature for one week, replacing the solution at least once after 24 hours or the next day; cut the tissue into 200 μm thick slices using a cryostat; transfer the sample to a gelatin-coated microscope slide containing solution C using a sample extractor (provided in the kit); remove excess solution from the slide with a pipette and blot dry with filter paper; allow the slices to air dry at room temperature and store in a slide box away from light until needed.

[0141] ③ Staining: Rinse the sections twice with double-distilled water for 4 minutes each time; place the sections in a mixture of double-distilled water with a volume ratio of 1:1:2 for 10 minutes; rinse the sections four times with distilled water for 4 minutes each time; counterstain the sections with crystal violet.

[0142] ④ Dehydration: Dehydrate the sections in 50%, 75%, and 95% ethanol for 4 minutes at each concentration gradient; dehydrate the sections in wastewater ethanol 4 times for 4 minutes each time.

[0143] ⑤ Clear the solution in xylene three times, for four minutes each time, and then seal the coverslip with a resin sealing medium.

[0144] Experimental results

[0145] Effects of the combination of Siegesbeckia orientalis and Astragalus membranaceus on the structural parameters of dendrites and dendritic spines in the hippocampus of CHF rats.

[0146] Table 6 Dendritic parameters of the hippocampus in rats of each experimental group

[0147]

[0148] Note: * P<0.05, ** P<0.01 vs. Sham group # P<0.05, ## P<0.01 vs. CHF group, n=3.

[0149] Figure 6 A is a schematic diagram of the intersection points of dendrites and concentric circles. As shown in Table 6, compared to the Sham group, the CHF group had a higher total number of intersection points (…). Figure 6 B and C), number of branches ( Figure 6 D) and branch length ( Figure 6E) was significantly reduced (P < 0.05); compared with the CHF group, the Siegesbeckia orientalis composition (high dose) of Example 1 of this invention increased the total number of intersections, the number of branches, and the branch length (P < 0.05). This result indicates that the Siegesbeckia orientalis composition can significantly promote dendritic growth in the hippocampus of rats with cognitive impairment after CHF.

[0150] Table 7. Parameters of dendritic spines in the hippocampus of rats in each experimental group.

[0151]

[0152] Note: * P<0.05, ** P<0.01 vs. Sham group # P<0.05, ## P<0.01 vs. CHF group, n=3.

[0153] Figure 7 A shows the dendritic spine characteristics of each group. Table 7 shows that, compared to the Sham group, the CHF group has a higher total number of dendritic spines (…). Figure 7 B) Number of mature dendritic thorns ( Figure 7 C) was significantly reduced (P < 0.05), and the number of immature dendritic spines ( Figure 7 D) also showed a decreasing trend; compared with the CHF group, the Siegesbeckia orientalis composition (high dose) of Example 1 of this invention could increase the total number of dendritic spines and the number of mature dendritic spines (P < 0.05). This result indicates that the Siegesbeckia orientalis composition can significantly promote the growth of dendritic spines in the hippocampus of rats with cognitive impairment after CHF.

[0154] Experiments have shown that the Siegesbeckia orientalis and Astragalus membranaceus compositions of Examples 2 and 3 can improve spatial memory and working memory in CHF rats, and improve cognitive function; inhibit the release of TNF-α and IL-1β in the hippocampus of CHF rats, and increase the content of IL-10 and BDNF; reduce the loss and damage of Nissl bodies in the brain tissue of CHF rats, improve the ultrastructure of neurons in the hippocampus of CHF rats, regulate synaptic plasticity, and promote the growth of dendrites and dendritic spines, similar to the Siegesbeckia orientalis and Astragalus membranaceus composition of Example 1.

Claims

1. The application of the Siegesbeckia orientalis and Astragalus membranaceus combination in the preparation of drugs to improve cognitive impairment after heart failure, characterized by: The composition of the hengjiaqin consists of astragalus extract, hengshengca extract and xiangjiapi extract; the mass ratio of the astragalus extract, hengshengca extract and xiangjiapi extract is (17-19):(2-4):1.

Images