Application of gynostemma pentaphyllum polysaccharide to prevention of vascular wall thickening caused by space microgravity and medicine

Abstract

The invention discloses application of gynostemma pentaphyllum polysaccharide to prevention of vascular wall thickening caused by space microgravity and a medicine, the gynostemma pentaphyllum polysaccharide is applied to prevention of vascular wall thickening caused by space microgravity and the medicine for prevention of vascular wall thickening caused by space microgravity, and the medicine contains the gynostemma pentaphyllum polysaccharide; the gynostemma pentaphyllum polysaccharide reduces thickening of blood vessel walls by inhibiting proliferation and migration of vascular smooth muscle cells, can be used for preventing or treating aorta blood vessel wall thickening caused by space microgravity, and is suitable for health guarantee of astronauts in long-term space missions.

Description

Technical Field

[0001] This invention belongs to the field of disease treatment caused by microgravity, and particularly relates to Gynostemma pentaphyllum polysaccharide for the prevention of blood vessel wall thickening caused by microgravity and related drugs. Background Technology

[0002] Spaceflight experiments have shown that the microgravity environment in space has various physiological effects on the human body. For example, microgravity can induce adaptive changes in the cardiovascular system, leading to cardiovascular dysfunction, aortic wall thickening, and fluid reversal, with aortic wall thickening being a significant issue. Prolonged exposure to microgravity alters the structure and function of blood vessel walls, resulting in thickening and consequently affecting the normal function of the cardiovascular system. Currently, treatment options for aortic wall thickening caused by microgravity are limited, thus necessitating the development of effective therapeutic drugs.

[0003] In recent years, drug research targeting cardiovascular diseases induced by microgravity has focused on improving myocardial metabolism, enhancing contractility, and regulating vascular function. For example, positive inotropic drugs such as milrinone (a phosphodiesterase inhibitor) and levosimendan improve cardiac function by enhancing myocardial contractility and are suitable for refractory heart failure; coenzyme Q10 and trimetazidine optimize myocardial energy metabolism and reduce oxidative damage caused by microgravity; vasoactive drugs such as ACEI / ARBs (e.g., captopril, losartan) and calcium channel blockers (e.g., amlodipine) can regulate vascular tone and alleviate blood flow abnormalities in microgravity environments; and novel targeted drugs based on the TRIM25 signaling pathway and autophagy mechanism discovered in space experiments are being developed to target immune regulation and cell repair.

[0004] The thickening of the aortic wall caused by microgravity is mainly manifested as an increase in the volume and weight of myocardial cells, especially the thickening of the left ventricular wall, increased myocardial fibrosis, and decreased elasticity of the aortic wall. It is also accompanied by arrhythmias and may lead to complications such as orthostatic hypotension or hypertension, abnormal lipid metabolism, skeletal muscle atrophy, and bone loss.

[0005] Gynostemma pentaphyllum (Thunb.) Makino, GPP, is a perennial herbaceous vine belonging to the genus Gynostemma in the family Cucurbitaceae. It is a plant used both as food and medicine. The active ingredients of Gynostemma pentaphyllum are macromolecules such as polysaccharides, flavonoids, and saponins, which have detoxifying, anti-inflammatory, invigorating, and fat-reducing effects. It is widely used in the treatment and prevention of hyperlipidemia, hypertension, obesity, insomnia, and chronic inflammation. Summary of the Invention

[0006] The purpose of this invention is to provide Gynostemma pentaphyllum polysaccharide for preventing blood vessel wall thickening caused by microgravity in space, thereby solving the problem of blood vessel wall thickening caused by microgravity in space.

[0007] The present invention adopts the following technical solution: Gynostemma pentaphyllum polysaccharide is used to prevent blood vessel wall thickening caused by microgravity in space.

[0008] The present invention also adopts the following technical solution: a drug for preventing thickening of blood vessel walls caused by microgravity in space, the drug containing Gynostemma pentaphyllum polysaccharide.

[0009] The present invention also adopts the following technical solution: a drug package, comprising a drug for preventing thickening of blood vessel walls caused by microgravity and an instruction manual describing the drug for therapeutic use.

[0010] The beneficial effects of this invention are: In this invention, Gynostemma pentaphyllum polysaccharide reduces the thickening of the blood vessel wall by inhibiting the proliferation and migration of vascular smooth muscle cells. It can be used to prevent or treat aortic thickening caused by microgravity in space and is suitable for the health protection of astronauts during long-term space missions. Attached Figure Description

[0011] Figure 1 A, C, and E represent the overall results of HE staining of the thoracic aorta in the CON, HU, and GPP groups, respectively; B, D, and F represent the overall results of HE staining of the thoracic aorta in the CON, HU, and GPP groups, respectively; G represents the data analysis results of the CON, HU, and GPP groups; H represents the rat tail suspension model simulating microgravity with a head-down (-30°) position (HU). Figure 2 A, B, and C represent A7r5 cells cultured under normal gravity conditions for 48, 72, and 96 h, respectively; D, E, and F represent A7r5 cells cultured under simulated microgravity conditions in a two-dimensional clinostat system for 48, 72, and 96 h, respectively; (G) represents the A7r5 cell viability of AF as detected by the CCK-8 assay. The data points represent the absorbance values ​​at 450 nm measured under standard culture conditions after incubation with WST-8® reagent for 30 min. Error bars represent the mean ± standard deviation (n=16). Statistical significance was determined by an unpaired t-test; H represents the two-dimensional clinostat system. Figure 3 A shows the H2S content in the serum of rats in the CON, HU, and GPP groups; B shows the transcriptomic analysis results of the rat aorta. Figure 4 Results of short-chain fatty acid content in the feces of rats in the CON, HU, and GPP groups. Detailed Implementation

[0012] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0013] This invention discloses the use of Gynostemma pentaphyllum polysaccharide to prevent blood vessel wall thickening caused by microgravity in space.

[0014] The Gynostemma pentaphyllum polysaccharide is used to inhibit the generation of H2S signaling molecules in blood vessels, thereby inhibiting the thickening of blood vessel walls caused by microgravity.

[0015] The Gynostemma pentaphyllum polysaccharide is used to inhibit the production of H2S signaling molecules in vascular smooth muscle cells, thereby inhibiting the thickening of the blood vessel wall caused by microgravity.

[0016] The Gynostemma pentaphyllum polysaccharide is used to inhibit the production of H2S signaling molecules in aortic vascular smooth muscle cells, thereby inhibiting the thickening of the vascular wall caused by microgravity.

[0017] The Gynostemma pentaphyllum polysaccharide is used to decompose and produce short-chain fatty acids, which in turn act on desulfurized Vibrio, thereby reducing the release of H2S by the desulfurized Vibrio and thus reducing the cell proliferation rate.

[0018] The H2S is used to accelerate the proliferation rate of A7r5 cells.

[0019] The present invention also discloses a drug for preventing blood vessel wall thickening caused by microgravity in space, which contains Gynostemma pentaphyllum polysaccharide.

[0020] The present invention also discloses a drug packaging comprising a drug for preventing thickening of blood vessel walls caused by microgravity and an instruction manual describing the drug for therapeutic use.

[0021] Example In this embodiment, SPF-grade rats were purchased from the Experimental Animal Center of Xi'an Jiaotong University and passed the medical and experimental animal ethics review of Northwestern Polytechnical University.

[0022] Eighteen male rats of similar weight (200-220 g) were randomly divided into a control group (CON group), a treatment group (GPP group), and a tail suspension group (HU group). They were housed in a standard SPF-grade animal facility under a temperature (22±2 ℃), humidity (50±10 %), and a 12-hour light / dark cycle. The CON group consisted of 7 rats, the GPP group of 6 rats, and the HU group of 5 rats. The CON group received normal daily feeding without any treatment, but were administered an equal volume of physiological saline daily by gavage (as a volume control). The GPP group rats were tail-suspended and administered 300 mg of Gynostemma pentaphyllum polysaccharide dissolved in physiological saline daily by gavage. The HU group was tail-suspended to simulate weightlessness, and was administered the same amount of physiological saline daily by gavage as the CON group.

[0023] The HU group underwent tail suspension treatment. A tail suspension device was used to fix the rats' tails, lifting their hind limbs off the ground while allowing their forelimbs free movement. Figure 1 As shown in (H), the suspension angle is approximately 30° to ensure the rats can eat and drink normally. The tail suspension is maintained for 28 consecutive days, with daily checks to ensure the suspension device is secure and to prevent tail injury.

[0024] Gavage was administered daily at fixed times. The GPP group received 300 mg of Gynostemma pentaphyllum polysaccharide dissolved in physiological saline via gavage, while the CON and HU groups received an equal volume of physiological saline via gavage. This continued for 28 days.

[0025] Twenty-eight days later, the rats were injected intraperitoneally with 20% urethane anesthetic to confirm complete anesthesia before sampling.

[0026] Samples were taken for HE staining and slide preparation. The slides were photographed using the Zhiying Medical Digital Slide Scanning System Science Edition, and the slide data was analyzed using ImageJ.

[0027] The results are shown in Table 1 and Figure 1 As shown in (AF), the intima-media thickness (IMT) of the thoracic aorta was significantly thickened in the HU group rats, while the IMT thickening induced by HU was counteracted in the GPP group rats. Figure 1 As shown in (G), the IMT layer thickness of rats in the GPP group was similar to that in the CON group, while the IMT layer thickness of rats in the HU group was higher than that in the CON and GPP groups.

[0028] Table 1 In this embodiment, rat aortic smooth muscle cells (A7r5, ATCC CRL-1444, RRID: CVCL_0137) were cultured in high-glucose DMEM medium (37 ℃ / 5% CO2) containing 10% fetal bovine serum and 1% penicillin-streptomycin. In the simulated microgravity experiment, cells from passages 3 to 5 were cultured at a density of 1×10⁻⁶ cells / mL. 4 One well per well was inoculated into a 24-well plate, sealed with silicone plugs, and then placed in a two-dimensional rotary table (Genintech, Suzhou, China) and run at 10 rpm. Figure 2 As shown in (H), the static control group was cultured under the same conditions but without rotation.

[0029] Inoculated into 24-well plates (1×10⁶ per well) 4 (cells) or 96-well plate (2 × 10 cells per well) 3Cell viability was assessed using a microplate reader (number of cells per well). The procedure was as follows: First, the culture supernatant was aspirated, and the cells were gently washed once with pre-warmed PBS to remove any residual metabolites that might interfere with subsequent reactions. Then, fresh complete culture medium was added; 500 μL was added to each well of a 24-well plate, and 100 μL to each well of a 96-well plate. CCK-8 reagent (APExBIO, Boston, USA) was added to each well, equivalent to 10% of the total culture medium volume. The plates were incubated at 37 ℃ / 5% CO2 in a dark incubator for 30 minutes. The absorbance was then measured at 450 nm using a Biotek Synergy H1 microplate reader (Biotek, Vermont, USA).

[0030] Bright-field imaging with a 10x objective lens revealed that, compared to the normal gravity control group, the cell density in the HU group was significantly increased after 48, 72, and 96 hours of culture. Figure 2 (AF). Subsequent CCK-8 assays confirmed the microgravity-dependent proliferation enhancement effect, showing a proliferation acceleration of 1.3±0.11-fold, 1.5±0.07-fold, and 1.3±0.08-fold at the corresponding time points, as shown in Table 2 and... Figure 2 As shown in (G), the proliferation rate of A7r5 cells exposed to simulated microgravity for 48h, 72h and 96h was higher than that of the CON group.

[0031] Table 2 Endogenous H2S in A7r5 cells was detected using the H2S fluorescent probe SF7-AM. After 72 h of treatment under simulated microgravity (SMG) / normal gravity conditions, the cell culture medium was aspirated, and the cells were gently washed once with phosphate-buffered saline (PBS). Subsequently, the cells were placed in a solution containing 2.5 μmol·L⁻¹ H2S. -1 Cells were incubated in fresh complete SF7-AM (Ex / Em: 488 / 525 nm; TOPSCIENCE, Shanghai, China) medium at 37 °C for 30 min; then washed with PBS, replaced with fresh medium, and finally observed using a Zeiss Axio Observer fluorescence microscope (Zeiss, Jena, Germany).

[0032] The H2S content in rat serum was detected using the methylene blue method.

[0033] 1. Sample and reagent preparation for the methylene blue method Rat serum samples were collected and centrifuged at 3000 r / min for 10 min at 4 ℃ to remove precipitate impurities. The clear serum supernatant was then used for further processing.

[0034] A: 3.3 mM trichloroacetic acid solution.

[0035] B: 2.5 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dye.

[0036] C: A mixture of 0.85% zinc acetate and 3% sodium hydroxide by mass-volume ratio.

[0037] D: Na₂S standard solutions of 5, 10, 20, 30, 40, 50, and 100 μM.

[0038] E: Rat serum F: Colorimetric solution (a 1:1 mixture of solution A and solution B) The specific experimental steps are as follows: 100 μL of serum was thoroughly mixed with 100 μL of 0.85% (w / v) zinc acetate, and then the chromogenic reagent was added. The mixture was incubated at room temperature for 20 min, and the absorbance at 670 nm was measured using a microplate reader. A standard curve was plotted with different concentrations of Na₂S on the x-axis and OD₆70 on the x-axis. The H₂S concentration in the sample was calculated based on the standard curve.

[0039] The results are shown in Table 3 and Figure 3 As shown in (A), the H2S content in the serum of rats in the HU group was higher than that in the CON group. It can also be seen that the H2S content in the serum of rats in the GPP group was significantly lower than that in the HU group. Therefore, it can be seen that Gynostemma pentaphyllum polysaccharide can effectively resist the increase in H2S content in rat serum caused by simulated microgravity.

[0040] Table 3 Serum H2S levels in rats were measured, and cecal contents samples were simultaneously collected for transcriptomics analysis. ANOVA analysis is a commonly used method in bioinformatics, often used to compare differences in gene expression levels between different groups. The ANOVA results for transcriptomics are shown below. Figure 3 As shown in (B), the content of *Desulfovibrio* in the cecal contents of rats in the tail suspension group increased. *Desulfovibrio* converts sulfate to H2S in the body through dissimilatory sulfate reduction, and is one of the main biological sources of H2S in both natural and artificial environments.

[0041] from Figure 3As shown in (B), tail suspension treatment increases the abundance of Desulfuric Vibrio in rat feces, which in turn increases the source of hydrogen sulfide in rats and increases the H2S content in serum. This suggests that tail suspension increases the content of Desulfuric Vibrio in the rat intestine, leading to an increase in H2S content in serum. After administration, the H2S content in rat serum returns to normal levels. Therefore, it can be demonstrated that the increase in H2S leads to accelerated proliferation of A7r5 cells.

[0042] like Figure 4 As shown, tail suspension treatment led to a decrease in the content of short-chain fatty acids in rat feces. After intervention with Gynostemma pentaphyllum polysaccharide, the content of short-chain fatty acids in the Gynostemma pentaphyllum group rats returned to the level of the CON group, effectively counteracting the increase in short-chain fatty acid content caused by tail suspension, and ultimately counteracting the thickening of the intima-media layer (IMT) in rats caused by tail suspension. Therefore, this example demonstrates that Gynostemma pentaphyllum polysaccharide can effectively counteract the thickening of the intima-media layer (IMT) in rat arteries caused by tail suspension.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Gynostemma pentaphyllum polysaccharide is used to prevent thickening of blood vessel walls caused by microgravity in space.

2. The Gynostemma pentaphyllum polysaccharide according to claim 1 for preventing blood vessel wall thickening caused by microgravity in space, characterized in that, The Gynostemma pentaphyllum polysaccharide is used to inhibit the generation of H2S signaling molecules in blood vessels, thereby inhibiting the thickening of the blood vessel wall caused by microgravity.

3. The Gynostemma pentaphyllum polysaccharide according to claim 2 for preventing blood vessel wall thickening caused by microgravity in space, characterized in that, The Gynostemma pentaphyllum polysaccharide is used to inhibit the production of H2S signaling molecules in vascular smooth muscle cells, thereby inhibiting the thickening of the blood vessel wall caused by microgravity.

4. The Gynostemma pentaphyllum polysaccharide according to claim 3 for preventing blood vessel wall thickening caused by microgravity in space, characterized in that, The Gynostemma pentaphyllum polysaccharide is used to inhibit the production of H2S signaling molecules in aortic vascular smooth muscle cells, thereby inhibiting the thickening of the vascular wall caused by microgravity.

5. The Gynostemma pentaphyllum polysaccharide according to claim 4 for preventing blood vessel wall thickening caused by microgravity in space, characterized in that, The Gynostemma pentaphyllum polysaccharide is used to decompose and produce short-chain fatty acids, which in turn act on desulfurized Vibrio, thereby reducing the release of H2S by desulfurized Vibrio and thus reducing the cell proliferation rate.

6. The Gynostemma pentaphyllum polysaccharide according to claim 5 for preventing blood vessel wall thickening caused by microgravity in space, characterized in that, The H2S is used to accelerate the proliferation rate of A7r5 cells.

7. A drug for preventing blood vessel wall thickening caused by microgravity in space, characterized in that, This medicine contains Gynostemma pentaphyllum polysaccharide.

8. A drug packaging, characterized in that, Includes the medicament for preventing vascular wall thickening caused by microgravity in space as described in claim 7, and instructions for use of the medicament for treatment.

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