Application of mogroside in the preparation of drugs for preventing and treating intervertebral disc degenerative diseases

Abstract

The present invention provides an application of mogroside in the preparation of a medicament for preventing and treating intervertebral disc degenerative diseases; the examples of the present application prove that mogroside has antioxidant stress, anti-inflammatory and anti-aging effects on nucleus pulposus-derived mesenchymal stem cells (NPMSCs); mogroside can improve oxidative stress-induced cellular senescence in NPMSCs; in addition, in vivo experiments in a rat model show that mogroside can reduce the degradation of the extracellular matrix of NPMSCs, delay the process of intervertebral disc degeneration, and has the characteristics of mild action and few side effects.

Description

Technical Field

[0001] The present invention relates to the field of medical technologies for intervertebral disc diseases, and particularly relates to the application of grosvenorine in the preparation of drugs for treating and delaying intervertebral disc degenerative diseases. Background Art

[0002] Intervertebral disc degeneration (IDD) is an important pathological factor causing neck, waist, and back pain, and plays an important role among the main diseases affecting people's health in the world today. Generally, IDD is considered to be one of the most important initiating factors for intervertebral disc degenerative diseases such as neck, shoulder, waist, and leg pain. However, there is currently no therapeutic drug for IDD in clinical practice. Therefore, there is an urgent need to discover new drugs that can effectively treat IDD.

[0003] Current conventional treatment methods (conservative treatment or surgery) are only symptomatic treatments and are difficult to repair and reconstruct the structure and function of degenerated intervertebral discs. Biological treatment based on mesenchymal stem cell (MSC) transplantation shows good prospects in the treatment of IDD, but the transplanted MSCs are difficult to survive for a long time, which becomes the main obstacle to MSC biological treatment.

[0004] Grosvenorine (Gro) is a natural flavonoid compound widely present in Siraitia grosvenorii, and has antioxidant stress, anti-inflammatory, and anti-aging effects. At present, the application of grosvenorine in the preparation of drugs for treating and delaying intervertebral disc degenerative diseases has not been reported. Summary of the Invention

[0005] In view of the above problems, the present application provides the application of grosvenorine in reducing the damage caused by oxidative stress to NPMSCs and anti-aging of NPMSCs, thereby realizing the application of grosvenorine in the preparation of drugs for treating and delaying intervertebral disc degenerative diseases.

[0006] The technical solution provided by the present application is as follows:

[0007] First, the present application provides the application of grosvenorine in the preparation of drugs for preventing and treating intervertebral disc degenerative diseases. The molecular formula of the above-mentioned grosvenorine is: C 33 H 40 O 19 , the molecular weight is: 740.66, and the structural formula is shown in formula (Ⅰ):

[0008]

[0009] Furthermore, the above-mentioned intervertebral disc degenerative disease refers to the intervertebral disc degeneration caused by the decrease in the number and the decline in the function of nucleus pulposus-derived mesenchymal stem cells (NPMSCs).

[0010] Secondly, the present application provides the use of mogroside in the preparation of a drug for preventing and treating TBHP-induced oxidative stress injury of NPMSCs. The development of oxidative stress injury of NPMSCs will further lead to intervertebral disc degeneration.

[0011] Thirdly, the present application provides a pharmaceutical composition for treating and delaying intervertebral disc degenerative diseases. The active ingredient of the composition includes mogrol flavone, and its dosage forms include pharmaceutically acceptable dosage forms such as tablets, granules, emulsions or capsules.

[0012] Mogrol flavone is a natural flavonoid compound widely present in various plants. The present invention has determined that mogrol flavone can relieve the senescence of NPMSCS induced by tert-butyl hydroperoxide (TBHP), and has antioxidant stress and anti-inflammatory effects on endogenous nucleus pulposus-derived mesenchymal stem cells (NPMSCs). The examples of the present application have determined the anti-aging effect in vitro cell experiments and in vivo SD rat IDD animal models. Mogrol flavone can improve the senescence induced by oxidative stress in NPMSCs, and can delay intervertebral disc degeneration by reducing the degradation of the extracellular matrix of NPMSCs. Therefore, mogrol flavone can be regarded as a potential drug for treating intervertebral disc degenerative diseases; and compared with western medicines, it has the characteristics of mild action and small side effects. Description of the Drawings

[0013] Figure 1 It is a micrograph of NPMSCs;

[0014] Among them, A-C are primary NPMSCs, the third-generation NPMSCs (200 times), and the third-generation NPMSCs (400 times) in sequence.

[0015] Figure 2 It is the results of detecting the cell viability and cell proliferation by Cell Counting Kit-8 (CCK-8) after treating NPMSCs with different concentrations of mogrol flavone (Gro) for 6h, 12h and 24h.

[0016] Figure 3 It is the result of senescence-associated β-galactosidase (SA-β-Gal) staining.

[0017] Figure 4 It is the immunofluorescence detection result of detecting the expression of senescence-related protein (p16) in NPMSCs in different groups by immunofluorescence.

[0018] Figure 5 Results of the experiment on the alleviation of TBHP-induced senescence of NPMSCs by mogroside

[0019] Figure 6 Results of hematoxylin-eosin staining and safranin-fast green staining at 4 weeks after fine needle aspiration in different groups Detailed implementation manners

[0020] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings

[0021] The mogroside used in the examples was purchased from Ruijing Scientific Research Procurement Platform, (Cayman, USA), 34810 - 25mg

[0022] All the experiments involved in the following examples have been approved by the Ethics Committee of Subei People's Hospital Affiliated to Yangzhou University

[0023] Example 1

[0024] 1. Experimental procedures

[0025] (1) Isolation and culture of NPMSCs

[0026] Sprague-Dawley (SD) rats (age, 2 - 4 months; body weight, 200 - 300 g) were obtained from Yangzhou University (license number SYXK (Su) 2022 - 0044).

[0027] First, the nucleus pulposus tissue obtained from SD rats was carefully isolated under sterile conditions through a microscope, and then the nucleus pulposus tissue was mechanically broken into 1 mm 3 and digested with 0.2% type II collagenase (Gibco, USA) for 12 hours at 37 °C and 5% CO2. After filtering with a 0.75 μm cell filter and centrifuging at 1000 rpm for 3 minutes, the cells washed with phosphate buffer solution (PBS) were resuspended in a solution containing 10% fetal bovine serum (FBS, HyClone, USA) and 1% antibiotic (Gibco, USA), and then cultured at 37 °C and 5% CO2; when the NPMSCS reached 80% confluence, subculture was carried out at a ratio of 1:3. All experiments used the third-generation NPMSCs

[0028] (2) Cell treatment and cell viability determination

[0029] To establish an oxidative stress injury model of NPMSCs, in this application, NPMSCs were cultured in complete medium (high-glucose DMEM medium, Gibco, USA) containing TBHP (100 μM, Sigma-Aldric, USA) for 6 hours. Before adding TBHP (100 μM), the cells were pretreated with mogroside at different concentrations (0 - 800 μg / ml, 0 - 24 h) to study the appropriate dose and time of mogroside. According to the manufacturer's instructions, the cell counting kit kit-8 (CCK-8, Beyotime, China) was used to evaluate cell viability. Briefly, 2×10 3 cells / well were seeded in a 96-well plate and cultured overnight at 37 °C and 5% CO2. Then, a mixture of 10 μL of CCK8 reagent and 100 μL of complete medium was added to each well and incubated at 37 °C for 1 hour. The optical density (OD) value of each well was measured at 450 nm using an enzyme-linked immunosorbent assay reader (Bio-Rad, USA).

[0030] (3) Senescence-associated β-galactosidase (SA-β-Gal) staining

[0031] NPMSCs were seeded in a 6-well plate at a density of 4×10 5 cells / well at 37 °C and 5% CO2. SA-β-Gal staining was performed according to the instructions provided by the SA-β-Gal staining kit (Beyotime, China). The cells were fixed with SA-β-gal fixative at room temperature for 15 minutes, and then incubated overnight at 37 °C in the absence of CO2 with SA-β-Gal working solution. The cells were observed under a microscope and analyzed using ImageJ software.

[0032] Figure 3 Among them, the Control group was the control group without any treatment, and the addition amount of the BHP group was 100 μM. The addition amount of Gro in the Gro+TBHP group was 400 μg / ml, and the addition amount of TBHP in the TBHP group was 100 μM.

[0033] (4) Western blot analysis

[0034] Total protein of NPMSCS was extracted by whole cell lysis method (Keygen Biotech, China), and the protein concentration was calculated using a BCA protein assay kit (Beyotime, China). Each sample containing 30 μg of protein was separated by 10 - 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane (Millipore, USA). After blocking with 5% non-fat milk at room temperature for 2 hours, the membrane was incubated with the primary antibody overnight at 4°C. Then the membrane was incubated with a horseradish peroxidase (HRP)-labeled secondary antibody (1:5000; Abcam). Then the membrane was observed using an enhanced chemiluminescence system, and the protein expression was analyzed using ImageJ software. The primary antibodies used in this study were: P16 (Proteintech, China) (1:1000), P21 (Proteintech, China) (1:1000), and GAPDH (Servicebio, China) (1:1000).

[0035] (5) Histological analysis

[0036] A SD rat model of IDD induced by fine needle puncture was established. Fifteen male SD rats (aged 2 - 4 months, weighing 200 - 300 g) were randomly divided into 3 groups: Control group (n = 5), IDD group (n = 5), and Gro treatment group (n = 5). After anesthesia with sodium pentobarbital, the tail skin was disinfected with povidone iodine, and then the CoccyidiIVD (CO 6 - 7) was punctured with a 21G puncture needle, rotated 360°, stayed in the intervertebral disc for 30 s, and the depth was 5 mm. After the operation, the Gro group was treated with mogroside Gro (100 mg / kg, 5 mg / mL Gro was suspended in 0.5% CMC-Na solution), and the Control group (normal mice, without any intervention) and the IDD group (intervertebral disc degeneration model group) were intragastrically treated with 0.5% sodium carboxymethylcellulose solution for 4 weeks. After killing the rats by injecting an overdose of sodium pentobarbital, the tails were collected, and the specimens were fixed with 4% paraformaldehyde for 48 hours, decalcified with EDTA for 1 month, dehydrated with gradient alcohol, and embedded in paraffin. The samples were cut into 5 μm sections, and then stained with hematoxylin-eosin (HE) and safranin O-fast green.

[0037] (6) Immunofluorescence staining

[0038] After preparing in vitro diagnostic specimens, the specimens were cut into 5-μm sections using a cryostat (Leica, Wetzlar, Germany). The sections were then fixed in 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 for 15 minutes. Subsequently, the cells were blocked with 10% bovine serum albumin at room temperature for 1 hour and incubated overnight at 4°C with a 1:100 dilution of primary antibodies: collagen type II (ABclonal, China) and Aggrecan (ABclonal, China). Then, they were incubated with a 1:200 dilution of FITC- or Cy3-labeled secondary antibodies in the dark for 1 hour at room temperature. The sections were observed and analyzed using a fluorescence microscope and ImageJ software.

[0039] (7) Statistical analysis

[0040] All data were analyzed using Prism 8 (GraphPad, La Jolla). Data are presented as mean ± standard deviation. The t-test or analysis of variance (ANOVA) was used to analyze data for two or more independent groups. The Kruskal-Wallis H test was used to analyze histological scores. P < 0.05 was considered statistically significant.

[0041] 2. Experimental results

[0042] (1) Characteristics of NPMSCs

[0043] Spindle-shaped NPMSCs were successfully isolated and cultured from rat intervertebral discs as Figure 1 shown. Primary NPMSCs were slender and spindle-shaped, growing in a sunflower-like pattern; passage 3 NPMSCs grew in a fusiform shape.

[0044] (2) Effects of mogroside on the cell viability and proliferation ability of NPMSCs

[0045] The CCK-8 assay was used to evaluate the effects of Gro and TBHP on the viability of NPMSCs. As the results showed Figure 2 shown, Figure 2 the cell proliferation results after treating NPMSCs with different concentrations of Gro for 6 h, 12 h, and 24 h were statistically analyzed. It was found that there was no obvious cytotoxic effect at a Gro concentration of up to 400 μg / ml for 24 hours. Therefore, a concentration of 400 μg / ml was selected for the following experiments.

[0046] In addition, after treatment with TBHP, cell viability decreased in a dose-dependent manner. Therefore, 100 μM TBHP was used for the following experiments.

[0047] (3) Effects of mogroside on SA-β-Gal staining in NPMSCs

[0048] The accumulation of SA-β-Gal is one of the hallmarks of cellular senescence, and the SA-β-Gal kit stains senescent cells blue. The staining results are as Figure 3 shown Figure 3 follows. Among A-C, the staining results of the Control group, TBHP group, and Gro+TBHP group are shown in sequence. Senescent cells show high expression of SA-β-gal in blue staining. It can be seen that the positive rate of SA-β-Gal in senescent NPMSCs in the TBHP group is significantly higher than that in the Control group (P<0.05), while the number of SA-β-Cal positive cells in the Gro+TBHP group decreases.

[0049] (4) Mogroside inhibits oxidative stress-induced senescence of NPMSCs

[0050] Widespread features of cellular senescence are cell cycle arrest and the production of senescence-associated secretory phenotype (SASP). To investigate the protective effect of mogroside in TBHP-induced senescence of NPMSCS, Western blot and immunofluorescence staining were used to detect the expression of cell senescence-related proteins (p16 and p21).

[0051] Figure 4 The immunofluorescence detection results of the expression of senescence-related protein (p16) in NPMSCs in different groups by immunofluorescence are shown. Figure 4 Among them, A shows the expression results of senescence-related proteins p16 and p21 detected by Western Blot among groups; B shows the quantitative analysis results of the protein expression of p16 in different groups; C shows the quantitative analysis results of the protein expression of p21 in different groups.

[0052] Figure 5 The experimental results of mogroside alleviating TBHP-induced senescence of NPMSCs are shown. Among them, DAPI is nuclear staining, and Merge represents the combination of p16 and DAPI.

[0053] Figure 4 and Figure 5 Western blot and immunofluorescence staining show that Gro treatment significantly reduces the upregulation of p16 and p21 proteins induced by TBHP.

[0054] In summary, these results indicate that mogroside plays a protective role in TBHP-induced oxidative stress injury of NPMSCs. Mogroside can alleviate TBHP-induced senescence at the cellular level and improve disc degeneration in rats with fine needle puncture at the animal model level.

[0055] (5) Mogroside improves disc degeneration at the animal model level of SD rats

[0056] A rat model of intervertebral disc degeneration was established by fine needle puncture to study the effect of mogroside in vivo on intervertebral disc degeneration (IDD).

[0057] Figure 6 Results of hematoxylin-eosin staining ( Figure 6 shown in A) and safranin-fast green staining ( Figure 6 shown in B) at 4 weeks after fine needle puncture for different groups. Hematoxylin-eosin staining and safranin O-fast green staining showed that the nucleus pulposus tissue in the Control group occupied most of the space of the intervertebral disc, and the nucleus pulposus cells were well distributed in the matrix. In contrast, the nucleus pulposus tissue in the IDD group almost disappeared, and the boundary between the nucleus pulposus and the annulus fibrosus was severely damaged. However, Gro treatment alleviated the degeneration and morphological changes of the nucleus pulposus and the annulus fibrosus, indicating that mogroside improved the condition of IDD in SD rats in vivo.

[0058] The above experiments demonstrated that mogroside could alleviate the degree of intervertebral disc degeneration induced by fine needle puncture in SD rats.

[0059] One embodiment of the present invention has been described in detail above, but the above description is only a preferred embodiment of the present invention and cannot be considered as limiting the scope of implementation of the present invention. Therefore, equivalent changes and improvements made according to the claims of the present invention still fall within the scope covered by the present invention.

Claims

1. Use of mogroside in the preparation of a drug for preventing and treating intervertebral disc degenerative diseases; the intervertebral disc degenerative diseases refer to intervertebral disc degeneration caused by a decrease in the number and function of nucleus pulposus mesenchymal stem cells.

2. The application according to claim 1, characterized in that The dosage form of the drug includes one of tablets, granules, emulsions, and capsules.

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