Use of Qixuekang in preparing a medicine for improving senility

Abstract

The application provides a use of Qixuekang in preparation of a medicine for improving aging. Experimental researches prove that Qixuekang can significantly improve natural aging of mice, improve the exercise capacity and muscle function of old mice, and Qixuekang can improve the mitochondrial membrane potential, increase the expression of mitochondrial neogenesis related genes and reduce the production of active oxygen, improve the mitochondrial function of replicative senescent cells, and further improve the replicative senescence state of the natural aging cell line 2BS.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and particularly to the use of Qi Xue Kang in the preparation of drugs for improving aging. Background Technology

[0002] Aging is the process of functional decline in the body's tissues and organs as we age, and it is closely related to normal embryonic development, bodily aging, and age-related diseases. In recent years, research on cellular senescence has become increasingly in-depth, and its biological role in anti-aging has received growing attention. Studies have shown that cellular senescence refers to the gradual decline in normal physiological functions and proliferative capacity of cells over time or under external stress, leading to cell cycle degeneration. This process is significantly linked to tumors, tissue regeneration, and overall aging. Since the mitochondrial senescence theory was proposed, mitochondria have become a focus of aging research. With age, mitochondrial function gradually weakens, and cellular degeneration is caused by reactive oxygen species (ROS), with the mitochondrial senescence theory identifying mitochondria as the primary producer of ROS. There is a demand in this field for drugs that can improve aging. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides the use of Qi Xue Kang in the preparation of drugs for improving aging. Experimental studies have confirmed that Qi Xue Kang can improve aging in naturally aging mammals and enhance their muscle strength and / or motor abilities.

[0004] In a first aspect, the present invention provides the use of Qi Xue Kang preparations in the preparation of medicaments for improving aging.

[0005] As a specific implementation, the Qi and Blood Health Preparation of the present invention is used to improve the aging of naturally aging mammals.

[0006] As a specific implementation, the Qi Xue Kang preparation of the present invention improves the aging phenotype of replicative senescent cells, preferably, the cells are 2BS cells.

[0007] As a specific implementation scheme, the Qi Xue Kang preparation of the present invention treats and / or improves the mitochondrial function of senescent cells.

[0008] As a specific implementation scheme, the Qi and Blood Health Formula of the present invention slows down the aging of tissues and organs in naturally aging mammals.

[0009] As a specific implementation, the Qi and Blood Strengthening Preparation of the present invention improves the muscle strength and / or motor ability of naturally aging mammals.

[0010] Secondly, the present invention provides the use of Qi Xue Kang preparations in the preparation of medicaments for improving the senescent phenotype of replicative senescent cells.

[0011] As a specific implementation scheme, the cells are 2BS cells.

[0012] Thirdly, the present invention provides the use of Qi Xue Kang preparations in the preparation of medicaments for treating and / or improving mitochondrial function in senescent cells.

[0013] Fourthly, the present invention provides the use of Qi Xue Kang preparations in the preparation of medicaments for slowing down the aging of tissues and organs in naturally aging mammals.

[0014] Fifthly, the present invention provides the use of Qi Xue Kang preparations in the preparation of medicaments for improving muscle strength and / or motor ability in naturally aging mammals.

[0015] As a specific implementation of the above aspects, Qi Xue Kang preparation is the only active ingredient.

[0016] As a specific implementation of the above aspects, the Qi Xue Kang preparation also includes pharmaceutically acceptable excipients or carriers. The "pharmaceuticalally acceptable excipients / carriers" described in this invention refer to non-therapeutic active substances other than the active ingredient, and are excipients / carriers routinely used in the production of pharmaceuticals and formulation in the art.

[0017] As a specific implementation of the above aspects, the Qi Xue Kang preparation is an oral liquid, capsule, or lyophilized powder. The Qi Xue Kang preparation can also be other dosage forms of the Qi Xue Kang formula, such as other compound preparations based on the Qi Xue Kang formula, such as Qi Xue Kang dispersible granules, Qi Xue Kang water drop pills, Qi Xue Kang honey pills, etc.

[0018] As a specific implementation plan for the above aspects, in the Qi Xue Kang preparation, the Qi Xue Kang oral liquid is produced by Yunnan Baiyao Group Wenshan Qihua Co., Ltd., with the approval number Z53020831; the Qi Xue Kang freeze-dried powder is prepared as described in the specific embodiment.

[0019] Beneficial effects

[0020] Experimental studies have confirmed that Qi Xue Kang can significantly improve the aging of naturally aging mice, enhance the exercise capacity and muscle function of aged mice. In vitro mechanism studies have shown that Qi Xue Kang can increase mitochondrial membrane potential, increase the expression of mitochondrial regeneration-related genes and reduce reactive oxygen species production, improve mitochondrial function in replicative senescent cells, and thus improve the replicative senescence state of the naturally aging cell line 2BS. Attached Figure Description

[0021] Figure 1 This study demonstrates the effect of Qi Xue Kang on the vitality of young 2BS (A) and aged 2BS (B) cells.

[0022] Figure 2The images show SA-β-gal staining for cellular senescence (magnification × 400) (A), changes in the expression of IL1β and p21 mRNA, senescence-related secretory phenotypes, after drug administration (B), and changes in the expression of cellular senescence markers p21, p16, and p53 proteins after drug administration (C). This indicates that p < 0.05. This indicates that p < 0.01.

[0023] Figure 3 The graphs show mitochondrial membrane potential fluorescence staining (A), statistical quantitative bar chart (B), reactive oxygen species statistical quantitative bar chart (C), and mitochondrial regeneration-related gene expression statistical quantitative bar chart (D). This indicates that p < 0.05. This indicates that p < 0.01.

[0024] Figure 4 The appearance (A), muscle strength (B), and motor ability (C) of aged mice treated with Qi Xue Kang are shown.

[0025] Figure 5 The bar chart shows the statistical quantitative expression of serum IL-1β (A), MMP3 (B), and TNFα (C) in young normal mice, aged mice, and aged mice treated with Qi Xue Kang. This indicates that p < 0.01.

[0026] Figure 6 The image shows HE staining (A) and Masson staining (B) of heart, kidney, liver, and muscle tissues from young normal mice, aged mice, and aged mice treated with Qi Xue Kang, along with quantitative results (C). This indicates that p < 0.05. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0028] In the following examples, the Qi Xue Kang used in Example 1 is Qi Xue Kang freeze-dried powder, which is produced by Yunnan Baiyao Group Wenshan Qihua Co., Ltd. The specific preparation method is described in Example 1. The Qi Xue Kang used in Example 2 is Qi Xue Kang oral liquid, which is produced by Yunnan Baiyao Group Wenshan Qihua Co., Ltd., with approval number Z53020831.

[0029] Example 1 This experiment verified that Qi Xue Kang can inhibit 2BS replicative senescence and improve the senescence phenotype and mitochondrial function of replicative senescent 2BS cells.

[0030] 1. Experimental Materials 1) Experimental cells: Human diploid fibroblasts 2BS (China National Institutes for Food and Drug Control) 2) Experimental reagents: Premium fetal bovine serum (YiAoBang, 03.U16001DC), MEM cell culture medium (YiAoBang, 03.10002C-PS), 0.25% trypsin (YiAoBang, 03.13004A), tissue and cell RNA micro-extraction kit (Meiji Bio, R4012), StarScript III one-tube genomic de-generated reverse transcription premix (Kangrun Bio, A230), HieffUNICON® Universal Blue qPCR SYBR Green Master Mix (Yisheng Bio, 11184ES), p21, p16, p53, and actin primary antibodies (Wuhan Sanying, 10355-1-AP, 30519-1-AP, 10442-1-AP, 20536-1-AP), goat anti-rabbit secondary antibody (Zhongshan Jinqiao, ZB-2301). 3) Experimental drugs: Qi Xue Kang freeze-dried powder solution, prepared as follows: Take 170g of fresh Panax notoginseng, 20g of ginseng, and 14g of kudzu root, crush them into coarse powder, use 60% ethanol as solvent, impregnate and percolate, recover the ethanol, let stand and filter, concentrate the filtrate to a clear extract with a relative density of about 1.2, which is Qi Shen Ge clear extract; 100g of Astragalus membranaceus is decocted three times with water, 2.5 hours each time, filtered, the filtrate is concentrated to a clear extract with a relative density of about 1.20, ethanol is added, let stand and filter, the filtrate is concentrated to a clear extract with a relative density of about 1.20, which is Astragalus membranaceus clear extract; freeze-dry the Qi Shen Ge clear extract and Astragalus membranaceus clear extract separately, which are Qi Shen Ge dry extract powder and Astragalus membranaceus dry extract powder respectively. Mix 19.2g of Qi Shen Ge dry extract powder and 18.3g of Astragalus membranaceus dry extract powder evenly to obtain Qi Xue Kang freeze-dried powder. The Qi Xue Kang lyophilized powder contains the same amount of active ingredients as 1L of Qi Xue Kang oral liquid (excluding excipients). In cell experiments, a certain amount of Qi Xue Kang lyophilized powder was dissolved in 1mL of sterile PBS to obtain a Qi Xue Kang lyophilized powder solution. The content of active ingredients in this Qi Xue Kang lyophilized powder solution is equivalent to the content of active ingredients in 4mL of Qi Xue Kang oral liquid (excluding excipients).

[0031] 2. Experiment Content 1) Cell culture: Human diploid fibroblasts 2BS were cultured in MEM medium (10% FBS + 1% penicillin-streptomycin mixture) and cultured and passaged at 37 ℃ and 5% carbon dioxide.

[0032] 2) Construction and treatment of replicative senescent 2BS cell models: 2BS cells were passaged to passage 50, with a cell cycle of approximately one week, thus representing replicative senescent cells. Cells from passage 45 were considered young cells. Replicative senescent cells were treated with either Qi Xue Kang lyophilized powder solution (equivalent to 4 μL Qi Xue Kang lyophilized powder solution / mL culture medium) or PBS to establish an aged Qi Xue Kang group and an aged group, respectively. Similarly, young cells were treated with either Qi Xue Kang lyophilized powder solution (equivalent to 4 μL Qi Xue Kang lyophilized powder solution / mL culture medium) or PBS to establish a young Qi Xue Kang group and a young group, respectively. Cells were collected after 24 hours for subsequent experiments.

[0033] 3) Cell Viability Assay: Cell Counting Kit-8 is a WST-8-based cell viability assay kit. WST-8 is a compound similar to MTT, which, in the presence of an electron coupling reagent, can be reduced by some dehydrogenases in mitochondria to produce orange-yellow formazan. After adding 10% CCK8 reagent to each well of a 96-well plate, the plate was incubated at 37°C for 1 hour, and the absorbance was measured at 450 nm using a microplate reader. Each group had 6 replicates, and the absorbance was calculated according to the formula: .

[0034] 4) Senescence-related β-galactosidase (SA-β gal) staining (SABG staining method): The staining working solution is prepared according to the instructions of the "Cellular Senescence β-galactosidase Staining Kit". After shaking on a shaker for 30 minutes, it is filtered through a 0.22 μm microporous membrane. This method can effectively reduce the interference of crystallization of the staining working solution on image processing. The staining working solution should be protected from light and prepared fresh before use.

[0035] Procedure: Aspirate the culture medium from the culture dish, gently rinse three times with PBS for 5 min each time, then add quantitative β-galactosidase staining and fixation solution (1 mL for a 6-well plate, 100 μL for a 96-well plate), fix at room temperature for 15 min, aspirate the cell fixative, and wash the cells three times with PBS for 5 min each time. Aspirate the PBS, and add quantitative staining working solution to each well to completely cover the bottom of the culture dish (1 mL for a 6-well plate, 100 μL for a 96-well plate). Incubate overnight in a 37°C incubator (air). Seal the 6-well plate with sealing film to prevent evaporation, and then wrap the 6-well plate with aluminum foil to block light.

[0036] The next day, discard the staining working solution and add enough quantitative nuclear solid red staining solution to completely cover the bottom of the culture dish for counterstaining (nuclear solid red counterstaining makes the cell outline clearer), and let it stand for 7 minutes. Discard the staining working solution, add PBS, observe under a microscope, and take a 100x photograph to record the staining. The blue-stained positive cells are senescent cells.

[0037] 5) RNA extraction and qRT-PCR detection of inflammatory mediator gene expression: RNA Extraction: RNA extraction was performed according to the instructions of the Tissue Cell RNA Micro-Extraction Kit (MegBio). The 6-well cell culture plates were washed once with PBS, and 200 µL of RTL Lysis Buffer and 200 µL of RNABinding Buffer were added. The mixture was transferred to a purification column and centrifuged at 13000 rpm for 1 min. 350 µL of Buffer RW1 was added to the column and centrifuged at 13000 rpm for 1 min. 500 µL of Buffer RW2 (diluted with ethanol) was added to the column and centrifuged at 13000 rpm for 1 min. This process was repeated once. The column was transferred to a 1.5 mL centrifuge tube. 30 µL of RNase-Free Water was added to the center of the column membrane. The mixture was allowed to stand for 1 min. Centrifuged at 13000 rpm for 1 min. The resulting solution contained cellular RNA.

[0038] cDNA synthesis: Prepare the reverse transcription reaction system according to the following composition.

[0039] The reaction conditions are as follows:

[0040] The cDNA obtained from the reaction was used for subsequent qPCR experiments.

[0041] qPCR reaction: Dilute cDNA in deionized water at a ratio of 1:5 and mix thoroughly with a vortex mixer. Prepare the qPCR reaction mixture (20 µL) according to the following proportions:

[0042] The qPCR reaction program is set as follows:

[0043] 36b4 was used as an internal reference gene, and the relative quantification method for gene expression was adopted. Way.

[0044] 6) Western blot detection of fibrosis marker expression: Cell lysis: Wash cells twice with PBS in 6-well cell culture plates, add 100 µL of RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors) to each well, and grind the cells thoroughly on ice to ensure complete cell lysis.

[0045] Protein quantification: The protein quantification process was performed according to the instructions of the BCA protein concentration assay kit (Beyotime Biotechnology). A 25 mg / mL protein standard was diluted to a final concentration of 2 mg / mL. Simultaneously, BCA working solution was prepared at a ratio of 50:1. Protein standards were added to the standard wells of a 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 µL, respectively, and PBS buffer was added to bring the total to 20 µL. The protein sample to be tested was diluted 10-fold with PBS, and 20 µL was added to each well. 200 µL of BCA working solution was added to all wells, and the mixture was gently aspirated and incubated at 37°C for 20 h. The absorbance of each well was read at 562 nm using a microplate reader, a standard curve was plotted, and the protein concentration of the sample was calculated. Subsequently, 25 µL of 5×Loading Buffer was added to the protein lysis buffer, and the plate was boiled at 100°C for 10 min in a metal bath, then stored at -20°C.

[0046] Protein electrophoresis and transfer: Prepare a 10% separating gel and a 5% stacking gel, load 10 μg of sample into each well, and perform electrophoresis at a constant voltage of 80 V for about 2 h. Then transfer the protein to an NC membrane at a constant current of 200 mA for 80 min.

[0047] Antibody incubation: Target protein antibody diluted 1:2500, internal control protein antibody diluted 1:2500, incubated overnight at 4°C. Then wash the membrane 6 times with TBST, 5 min each time. Label with secondary antibody diluted 1:5000, incubated at room temperature for 1 h. Develop the membrane after washing with TBST.

[0048] Data analysis: The grayscale values ​​of the Western blotting results were statistically analyzed using ImageJ software.

[0049] 7) Mitochondrial membrane potential staining a) Preparation of JC1 staining solution: Prepare JC-1 staining working solution by mixing JC-1 (200×), ultrapure water, and JC-1 staining buffer (50×) in a ratio of 1:160:40; b) JC-1 staining: Cells were cultured in six-well plates. After rinsing with PBS, 1 mL of JC-1 staining working solution was added to each well and mixed thoroughly. The cells were incubated at 37°C for 20 minutes.

[0050] c) Washing: Prepare a washing solution by mixing JC-1 staining buffer (5×) and distilled water at a ratio of 1:4. After staining, wash twice with the washing solution and add 2 mL of cell culture medium to each well.

[0051] d) Taking photos: taking photos with a fluorescence microscope.

[0052] Data analysis: Fluorescence intensity was statistically analyzed using ImageJ software.

[0053] 8) Detection of reactive oxygen species (ROS) in cells a) DCFH-DA probe staining: Cells were cultured in 96-well dark plates. After rinsing with PBS, 0.1 mL of fresh culture medium containing DCFH-DA probe was added to each well, with a final concentration of 10 μmmol / L. The plates were then incubated at 37°C for 20 min.

[0054] b) Detection: Detection was performed using a fluorescent microplate reader with an excitation wavelength of 488nm and an emission wavelength of 525nm.

[0055] 3. Experimental Results The experimental results in this section demonstrate that Qi Xue Kang can improve cellular replication and senescence, and this beneficial effect is related to its ability to improve mitochondrial function. Qi Xue Kang has no significant cytotoxic effect on cells. Figure 1 Qi Xue Kang treatment reduced the proportion of β-galactosidase-positive replicative senescent 2BS cells. Figure 2 (A). Simultaneously, Qi Xue Kang treatment reduced the high expression of cellular senescence markers p21, p16, and p53 caused by replicative senescence, as well as the expression of the SASP gene (…). Figure 2 (B) This indicates that Qi Xue Kang can improve cellular replication and senescence. The JC-1 staining intensity in mitochondria of senescent cells is lower than that of young cells, while administration of Qi Xue Kang significantly restores the fluorescence intensity (B). Figure 3 (A). Meanwhile, the ROS staining intensity of senescent cells was significantly increased, while administration of Qi Xue Kang significantly reduced the staining intensity ( ). Figure 3 In addition, Qi Xue Kang significantly upregulated mitochondrial regeneration-related genes (Tfam and Mfn2). Figure 3 (D). In summary, the results indicate that Qi Xue Kang can significantly increase mitochondrial membrane potential, promote mitochondrial regeneration, enhance mitochondrial function, and reduce reactive oxygen species production.

[0056] Example 2 This experiment verified that Qi Xue Kang can improve the aged appearance of aged mice, improve their motor ability, delay the aging of the mouse's body and major organs, and promote the healthy lifespan of the mouse.

[0057] 1. Experimental Materials 1) Experimental animals: 18-month-old male C57 mice, weighing 25±5g; 8-week-old male C57 mice, weighing 20±5g, were purchased from the Laboratory Animal Department of Peking University School of Medicine. Temperature (21-25℃), humidity (60±5%), and light (12 / 12 h light / dark cycle) were controlled, and free access to food and water was provided.

[0058] 2) Experimental drug: Qi Xue Kang oral liquid, dosage 400 μL / animal.

[0059] 2. Experiment Content 1) Animal experimental protocol: Twenty 18-month-old male C57 mice were randomly divided into two groups. One group, the Qi Xue Kang treatment group, was administered 400 μL of Qi Xue Kang oral solution per mouse via gavage. The other group, the aged mouse control group, was administered an equal volume of physiological saline via gavage. A separate group of young, normal mice was also administered an equal volume of physiological saline via gavage. Treatment was administered for 7 consecutive days each month, followed by a 3-week break, for a total of 6 months. During treatment, grip strength and rotarod fatigue resistance tests were performed monthly. After 6 months, blood was collected from the orbital sinus of the mice for serum SASP ELISA testing. The mice were euthanized by cervical dislocation, and major organs were fixed for histopathological examination.

[0060] 2) Mouse fatigue endurance test: Before the formal experiment, two groups of aged mice were placed in a fatigue rotarod apparatus and rotated at a constant speed of 15 rpm for a pre-experiment to familiarize the mice with the environment. During the formal experiment, the fall time of the mice while moving on the fatigue rotarod apparatus was recorded. Each mouse was tested three times, and the average fall time was calculated.

[0061] 3) Mouse grip strength measurement: Two groups of aged mice were placed on the grid of a grip strength meter, with their trunks horizontal and only their forepaws allowed to remain attached to the grid before any measurements were taken. The mouse's tail was gently pulled back, ensuring the mouse gripped the top of the grid and its trunk remained horizontal, and the maximum grip strength value displayed on the screen was recorded. This process was repeated twice to obtain five grip strength measurements. The maximum grip strength before release was recorded. The average performance of the animals in each trial is expressed as the average of the two trials.

[0062] 4) Mouse serum SASP ELISA assay: Mouse whole blood was incubated at room temperature for 30 min, then centrifuged at 3000 rpm for 15 min at 4℃, and serum was collected. The levels of IL-1β, MMP3, and TNF-α were detected using the ELISA kit according to the manufacturer's instructions.

[0063] 5) Histopathological examination: Tissue is fixed, embedded, sectioned, and stained with HE and Masson staining.

[0064] 6) Statistical analysis: Image J was used to measure the cross-sectional area of ​​muscle fibers in HE-stained muscle tissue images, and Image J was used to measure the area of ​​the blue region stained with Masson's stain in kidney tissue. Statistical analysis was performed using GraphPad Prism 9.0. One-way ANOVA was used to test the significance of results from two or more groups, with P < 0.05 considered statistically significant.

[0065] 3. Experimental Results Older mice exhibited disheveled, dull fur with sparse hair and graying, while older mice treated with Qi Xue Kang had relatively smooth, shiny fur with almost no hair loss or graying. Figure 4(A). Treatment with Qi Xue Kang (a traditional Chinese medicine) improved grip strength and significantly increased fatigue tolerance in aged mice compared to untreated aged mice. Figure 4 (B and C). Serum levels of SASPIL-1β, MMP3, and TNF-α in aged mice were significantly higher than in young mice, while treatment with Qi Xue Kang significantly reduced serum SASP levels, indicating that Qi Xue Kang improves the aging phenotype in aged mice. Figure 5 (A, B, and C). Treatment with Qi Xue Kang improved myocardial cell hypertrophy and increased connective tissue between myocardial fibers in the heart of aged mice; increased senescent cells, inflammatory infiltration, fibrosis, and lipid deposition in the liver; and glomerular sclerosis, renal tubular atrophy, and interstitial fibrosis in the kidneys. Figure 6 (A and B in the original text). The above results indicate that Qi Xue Kang administration can significantly improve the aging phenotype and organ aging in aged mice, promoting healthy lifespan.

Claims

1. Use of Qi Xue Kang preparations in the preparation of drugs for improving aging.

2. The use according to claim 1, wherein, The Qi Xue Kang preparation improves the aging process in naturally aging mammals.

3. Use of Qi Xue Kang preparations in the preparation of drugs for improving the senescent phenotype of replicative senescent cells.

4. The use according to claim 3, wherein, The cells in question are 2BS cells.

5. Use of Qi Xue Kang preparations in the preparation of drugs for treating and / or improving mitochondrial function in senescent cells.

6. Use of Qi Xue Kang preparations in the preparation of drugs for slowing down the aging of tissues and organs in naturally aging mammals.

7. Use of Qi Xue Kang preparations in the preparation of medicines for improving muscle strength and / or motor ability in naturally aging mammals.

8. The use according to any one of claims 1-7, wherein, In the Qi Xue Kang preparation, Qi Xue Kang is the only active ingredient.

9. The use according to any one of claims 1-7, wherein, The Qi Xue Kang preparation also includes pharmaceutically acceptable excipients or carriers.

10. The use according to any one of claims 1-7, wherein, The Qi Xue Kang preparation is an oral liquid, capsule, or lyophilized powder.

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