Application of ginsenoside Rg2 in preparation of medicine for treating multiple sclerosis

Abstract

The invention is applicable to the technical field of biological medicine, and provides application of ginsenoside Rg2 in preparation of a medicine for treating multiple sclerosis, and the ginsenoside Rg2 plays a role in treating MS through the following mechanisms: reducing the expression level of proinflammatory factors, promoting differentiation of regulatory T cells and inhibiting infiltration of immune cells to a central nervous system; the activation of an NLRP3 signal channel is inhibited, and the pyroptosis of nerve cells is reduced; damage of an immune system to the myelin sheath is prevented, and the ratio of the myelin sheath deprivation area to the white matter area is reduced; expression of transcription factors of oligodendroglia cells is up-regulated, proliferation and differentiation of oligodendroglia precursor cells are promoted, and remyelination is promoted; and microglial cell activation is inhibited. The problems that an existing medicine is limited in curative effect, remarkable in side effect, only capable of delaying progress, limited in administration mode and the like are solved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of ginsenoside Rg2 in the preparation of drugs for the treatment of multiple sclerosis. Background Technology

[0002] Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Its pathogenesis is closely related to autoimmune abnormalities, neuroinflammatory damage, and impaired myelin regeneration. It is characterized by an abnormal attack of the myelin sheath by the immune system (demyelination), leading to impaired nerve signal transmission and subsequently causing various neurological dysfunctions. Its main pathological feature is the induction of an inflammatory response, promoting microglial activation and T-cell infiltration. Clinically, it mainly manifests as limb weakness, sensory abnormalities, cognitive impairment, and motor function decline, severely impacting patients' quality of life. Currently, the incidence of MS is increasing year by year, and it has become one of the leading neurological diseases causing disability in young adults.

[0003] Existing research indicates that the core pathology of MS lies in: (1) abnormal activation and infiltration of immune cells (such as T cells, B cells, and macrophages) into the central nervous system, releasing pro-inflammatory factors (such as TNF-α, IL-17, and IFN-γ) to trigger an inflammatory cascade; (2) oligodendrocyte damage leading to demyelination and impaired nerve signal transmission; and (3) irreversible damage to nerve axons, resulting in permanent neurological deficits. Therefore, an ideal MS treatment drug must possess multiple functions, including anti-inflammatory regulation, neuroprotection, and promotion of myelin regeneration.

[0004] Ginsenoside Rg2 is a tetracyclic triterpenoid saponin extracted from ginseng (chemical formula: C). 42 H 72 O 13 Existing research has only reported its anti-apoptotic effects, improvement of cognitive dysfunction (animal models), and anti-inflammatory effects. Furthermore, its non-toxicity and safety to humans are guaranteed, making its development into a clinical drug highly promising.

[0005] Current clinical treatments for MS are mainly divided into disease-modifying therapies (DMTs), symptomatic treatments, and immunosuppressants, but they have the following key limitations: Limited therapeutic effect: Existing drugs (such as fingolimod) mainly focus on immunosuppression, which cannot effectively solve the neurodegenerative problems in the middle and late stages of the disease, nor can they promote the repair of damaged myelin sheaths. About 30% of patients do not respond to existing DMTs, and long-term use can easily lead to a decline in efficacy.

[0006] Significant side effects and poor safety: Immunosuppressants (such as cyclophosphamide and methotrexate) can easily cause systemic immunosuppression, leading to infection, liver and kidney damage and hematological abnormalities. Some DMTs (such as nastatinumab) may induce fatal complications such as progressive multifocal leukoencephalopathy (PML).

[0007] Unable to achieve nerve repair: Existing drugs have not addressed the core pathological aspects of MS—neural axonal protection and myelin regeneration. They can only slow disease progression and cannot reverse existing neurological deficits.

[0008] Limited administration methods: Most drugs require subcutaneous injection or intravenous infusion, resulting in poor patient compliance; oral drugs (such as fingolimod) pose risks such as bradycardia and lung infection, and have a narrow applicable population.

[0009] In addition, there is limited research on the application of natural compounds in the treatment of MS. Existing natural drugs (such as curcumin and resveratrol) have problems such as low bioavailability and unclear mechanisms of action, making it difficult to translate them into clinical treatments. Summary of the Invention

[0010] The purpose of this invention is to provide the application of ginsenoside Rg2 in the preparation of drugs for the treatment of multiple sclerosis, in order to solve the problems mentioned in the background art.

[0011] To address the above problems, the present invention provides the application of ginsenoside Rg2 in the preparation of drugs for the treatment or prevention of multiple sclerosis.

[0012] Another objective of this invention is to provide the application of ginsenoside Rg2 in the preparation of a drug for inhibiting inflammation of the central nervous system. The ginsenoside Rg2 is used to reduce the expression level of pro-inflammatory factors, while promoting the differentiation of regulatory T cells and inhibiting the infiltration of immune cells into the central nervous system.

[0013] Another objective of this invention is to provide the application of ginsenoside Rg2 in the preparation of neuroprotective drugs, wherein ginsenoside Rg2 is used to inhibit the activation of the NLRP3 signaling pathway and reduce neuronal pyroptosis.

[0014] Another objective of this invention is to provide the application of ginsenoside Rg2 in the preparation of drugs that promote myelin regeneration.

[0015] Furthermore, the ginsenoside Rg2 is used to prevent the immune system from destroying the myelin sheath and reduce the ratio of the area of ​​myelin sheath loss to the area of ​​white matter.

[0016] Furthermore, the ginsenoside Rg2 is used to upregulate the expression of oligodendrocyte transcription factors, promote the proliferation and differentiation of oligodendrocyte precursor cells, and promote myelin regeneration.

[0017] Another object of the present invention is to provide the application of ginsenoside Rg2 in the preparation of a drug that inhibits microglial cell activation.

[0018] Another object of the present invention is to provide a medicament for treating or preventing multiple sclerosis, comprising a pharmaceutically acceptable carrier and ginsenoside Rg2.

[0019] Furthermore, the concentration of ginsenoside Rg2 is 20-80 μmol / L.

[0020] Furthermore, the extraction method of ginsenoside Rg2 includes the following steps: One or more of the roots, stems and leaves of plants in the genus Panax of the Araliaceae family are crushed and extracted by reflux at 80°C with a 70%-80% ethanol aqueous solution to obtain an extract. The above extract was concentrated under reduced pressure until no alcohol odor was detected, and then loaded onto a D101 macroporous resin column. It was eluted sequentially with ethanol aqueous solutions of 30%, 50%, and 70% (v / v), and the eluent of the 70% ethanol aqueous solution was collected. The above eluent was purified by high performance liquid chromatography (HPLC) using a C18 column, a mobile phase of acetonitrile-water = 25:75, a flow rate of 1.0 mL / min, and a detection wavelength of 203 nm. The chromatographic peaks with retention times of 12.5–13.5 min were collected and then freeze-dried to obtain ginsenoside Rg2.

[0021] This invention addresses the technological bottlenecks and clinical needs in the current treatment of multiple sclerosis (MS). Through the novel application of the natural compound ginsenoside Rg2, it solves problems such as limited efficacy, significant side effects, only slowing disease progression, and restricted administration methods associated with existing drugs. Ginsenoside Rg2 exerts its therapeutic effect on MS primarily through the following mechanisms: 1. Improves mouse behavior: Reduces clinical symptoms in mice, significantly alleviates symptoms, and almost eliminates bilateral hind limb paralysis accompanied by forelimb paralysis.

[0022] 2. Improved motor function: In the rotarod experiment, mice treated with ginsenoside Rg2 stayed on the rod for a longer time, showing better motor ability.

[0023] 3. Anti-inflammatory regulation: It reduces the expression levels of pro-inflammatory factors (IL-1β, IL-18), while promoting the differentiation of regulatory T cells (Treg) and inhibiting the infiltration of immune cells into the CNS.

[0024] 4. Neuroprotection: Inhibits activation of the NLRP3 signaling pathway and reduces neuronal pyroptosis.

[0025] 5. Reduces myelin loss in spinal cord white matter: Ginsenoside Rg2 prevents the immune system from destroying the myelin sheath and reduces the ratio of myelin loss area to white matter area.

[0026] 6. Promotes myelin regeneration: Myelin regeneration refers to the process in multiple sclerosis (MS) where new oligodendrocytes wrap around exposed axons, forming a new myelin sheath, thereby partially or completely restoring the speed of nerve signal conduction and protecting the axon. Ginsenoside Rg2 can upregulate the expression of oligodendrocyte transcription factors (Olig2, SOX10), promote the proliferation and differentiation of oligodendrocyte precursor cells (OPCs), accelerate myelin regeneration, and repair demyelinating lesions.

[0027] 7. Inhibit excessive activation of microglia: In MS lesions, abnormal activation of microglia releases a large number of inflammatory mediators. Ginsenoside Rg2 can reduce the number of activated microglia. Attached Figure Description

[0028] Figure 1 The figure shows the effect of ginsenoside Rg2 on the clinical scores of EAE mice.

[0029] Figure 2 The figure shows the effect of ginsenoside Rg2 on the motor ability of EAE mice.

[0030] Figure 3 The figure shows the effect of ginsenoside Rg2 on the content of the inflammatory factor IL-1β in the spinal cord of EAE mice.

[0031] Figure 4 The figure shows the effect of ginsenoside Rg2 on the activation of microglia in the spinal cord tissue of EAE mice.

[0032] Figure 5 The figure shows the effect of ginsenoside Rg2 on demyelination of the white matter in the EAE spinal cord.

[0033] Figure 6 The figure shows the effect of human saponin Rg2 on the expression of the oligodendrocyte marker O4+.

[0034] Figure 7 The figure shows the effect of ginsenoside Rg2 on the expression of myelin MBP. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0036] In one embodiment of the present invention, a novel use of the natural compound ginsenoside Rg2 in the treatment of multiple sclerosis is discovered for the first time; wherein, the chemical name of ginsenoside Rg2 is 6-O-[α-L-pyranorhamnosyl-(1-2)-β-D-pyranoglycosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol, and the molecular formula is C 42 H 72 O 13 Ginsenoside Rg2 is derived from the roots, stems, or leaves of plants in the genus Panax (such as Panax ginseng CA Mey. and Panax quinquefolius L.) of the Araliaceae family, and can be prepared through natural extraction or semi-synthetic methods.

[0037] In a preferred embodiment of the present invention, a method for extracting ginsenoside Rg2 is also provided, specifically including the following steps: S1. Pulverize the root, stem or leaf of ginseng and extract it three times by reflux at 80°C with 70%-80% ethanol aqueous solution for 2 hours each time, and combine the extracts. S2. Concentrate the above extract under reduced pressure until there is no alcohol odor, then load it onto a D101 macroporous resin column and elute with ethanol aqueous solution with volume concentrations of 30%, 50%, and 70% in sequence. Collect the eluent of 70% ethanol aqueous solution to obtain the eluent. S3. The above eluent was purified by high performance liquid chromatography (HPLC) (chromatographic column: C18 column, mobile phase: acetonitrile-water = 25:75, flow rate 1.0 mL / min, detection wavelength 203 nm), and the chromatographic peak with retention time of 12.5-13.5 min was collected. After freeze drying, ginsenoside Rg2 monomer with a purity ≥98% was obtained.

[0038] This invention discloses for the first time a novel application of ginsenoside Rg2 in the treatment of multiple sclerosis (MS). This represents an unprecedented pharmaceutical application for this compound. Ginsenoside Rg2 can be used to prevent, treat, and / or alleviate MS and its symptoms, filling a gap in the field of natural compounds for MS treatment. Specifically, ginsenoside Rg2 simultaneously achieves anti-inflammatory, neuroprotective, and myelin regeneration effects, overcoming the limitations of existing single-target technologies. Its efficacy is significant: in the MS animal model (EAE), ginsenoside Rg2 not only significantly delays the onset of disease and reduces clinical scores, but also pathologically demonstrates its powerful repair ability to reduce demyelination and promote myelin regeneration, with effects equivalent to some positive control drugs (such as fingolimod), and exhibiting unique advantages in promoting repair. Ginsenoside Rg2 has high safety: as a natural product, its safety is superior to many synthetic immunosuppressants, with fewer potential side effects. The embodiments of the present invention provide novel potential treatment options for MS patients who do not respond to existing immunotherapies or have entered the progressive stage, especially addressing the unmet clinical need for nerve repair, and also provide new lead compounds and research ideas for the development of neuroimmunological drugs based on natural products.

[0039] Example 1, Experimental autoimmune encephalomyelitis (EAE) model and ginsenoside Rg2 treatment: 6-8 week old female C57BL / 6 mice were randomly divided into: (1) Model control group (Vehicle, saline), i.e., the Vehicle group in the attached figure; (2) Positive drug control group (Fingolimod FTY720, 1 mg / kg / day), i.e., the FTY720 group in the attached figure; (3) Ginsenoside Rg2 treatment group (G-Rg2, 30 mg / kg / day), i.e., the G-Rg2 group in the attached figure; 10 mice in each group were used to establish an experimental autoimmune encephalomyelitis (EAE) mouse model to simulate the symptoms of clinical MS. The specific method was: 200 μg of myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55) containing 4 mg / mL Mycobacterium tuberculosis was injected subcutaneously in Freund's adjuvant (CFA) for immunization. In addition, 200 ng of pertussis toxin was administered intraperitoneally (ip) on the day of immunization and 48 hours after immunization.

[0040] Starting from day 3 post-immunization, mice in the model control group were orally administered saline, mice in the positive control group were orally treated with fingolimod (at a dose of 1 mg / kg), and mice in the ginsenoside Rg2 treatment group were orally treated with ginsenoside Rg2 (at a dose of 30 mg / kg), for 27 consecutive days. Clinical scores of mice in each group were observed and recorded daily from the day of immunization. The clinical score used an internationally recognized 5-point scoring system: 0 points for no clinical symptoms; 1 point for loss of tail tension and mild clumsiness; 2 points for unilateral hind limb weakness that recovers after passive turning; 3 points for bilateral hind limb paralysis that does not recover after passive turning but can be moved after stimulation; 4 points for bilateral hind limb paralysis with forelimb paralysis; and 5 points for a near-death state or death.

[0041] On day 30 post-immunization, mice underwent a rotarod test. Specifically, mice were placed on an accelerator where the rotarod was gradually accelerated from 5 rpm to 50 rpm over 5 minutes. The time it took for the mouse to fall was recorded as the latency period. Each mouse was tested three times, and the average latency period was analyzed.

[0042] The therapeutic effect of ginsenoside Rg2 on EAE mice was detected using the above experimental methods. The detection indicators included clinical score and motor function. The results are as follows: Figure 1 and Figure 2 As shown.

[0043] from Figure 1 and Figure 2 It can be seen that compared with the model control group, the onset time of ginsenoside Rg2 treatment in mice was significantly delayed, and the highest clinical score was significantly lower. The highest clinical score in the model control group was 3.8±0.5, while the highest score in the ginsenoside Rg2 treatment group was 1.5±0.3 (P<0.01 vs. model control group). Throughout the experimental period, the average clinical score was comparable to that of the positive control group, and both were significantly lower than that of the model control group. In the rotarod test, the rotarod drop latency in the model control group was 227.9±14.32 (s), while the rotarod drop latency in the ginsenoside Rg2 treatment group was 283.8±7.09 (s), comparable to that of the positive control group, and both were significantly lower than that of the model control group (P<0.01 vs. model control group).

[0044] Example 2: Inflammatory Regulation of Ginsenoside Rg2 in EAE Mice: After the rotarod experiment described above, mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital solution. Following anesthesia, the heart was perfused with physiological saline until the liver turned white. The spinal cord was then rapidly dissected on ice. The spinal cord tissue was homogenized and used for ELISA (inflammatory factor level) detection. Spinal cord proteins were extracted and frozen at -80℃ for Western blot (protein expression level) detection.

[0045] The ELISA (inflammatory factor level) test specifically uses a commercial reagent kit and is performed according to the instructions to detect the level of a certain substance in the body.

[0046] The inflammatory regulatory effect of ginsenoside Rg2 on EAE mice was detected using the above experimental methods. The detection indicators were: the content of inflammatory factor IL-1β in mouse spinal cord tissue and the activation status of microglia.

[0047] Test results as follows Figure 3 and Figure 4 As shown: The IL-1β level in the model control group mice was 8.1±0.44 (pg / mL pro), while the IL-1β level in the ginsenoside Rg2 treatment group mice was 5.6±0.2 (pg / mL pro) (P<0.01 vs model control group). Throughout the experimental period, their IL-1β levels were comparable to those in the positive control group, both significantly lower than those in the model control group. The number of activated microglia in the model control group was 29.7±2.2, while the number of activated microglia in the ginsenoside Rg2 treatment group mice was 12±1.1. Throughout the experimental period, their activated cell numbers were comparable to those in the positive control group, both significantly lower than those in the model control group (P<0.01 vs model control group).

[0048] The above experimental method was used to detect the effect of ginsenoside Rg2 on the loss of myelin sheath in the spinal cord white matter. The detection index was myelin staining.

[0049] Test results as follows Figure 5 As shown: In the model control group, large areas of myelin sheath loss were observed in the spinal cord white matter, with the ratio of the area of ​​myelin loss to the area of ​​white matter being 23.45±3.32. In contrast, the ratio in the ginsenoside Rg2 group was 14.22±3.11, with a significantly reduced area of ​​myelin loss (P<0.01), and the effect was comparable to that in the positive drug control group mice.

[0050] Example 3: In vitro cell experiments to verify the myelin regeneration-promoting effect of ginsenoside Rg2: Oligodendrocyte precursor cells (OPCs) were cultured. Oligodendrocyte precursor cells (OPCs) from the cerebral cortex of newborn mice were isolated and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture. The cells were maintained at 37°C and 98% relative humidity with 5% CO2. The OPCs were divided into a model control group (no drug) and ginsenoside Rg2 groups (20μM, 40μM, and 80μM).

[0051] Immunofluorescence assays were performed on all groups. Cells were pretreated with ginsenoside Rg2 (20 μM, 40 μM, 80 μM) for 7 days, with the corresponding drug concentration culture medium replaced every 2 days to ensure stable drug concentration. After 7 days of culture, the culture medium in each well was discarded, and the cells were gently washed 3 times (5 minutes each time) with pre-cooled PBS buffer. 1 mL of 4% paraformaldehyde was added to each well for fixation, and 1 mL of 5% BSA blocking solution was added to each well. After blocking at 37°C for 1 hour, primary antibodies (O4+ antibody and MBP antibody) were added, and the cells were incubated overnight at 4°C. The next day, cells were incubated with secondary antibodies and observed under a fluorescence microscope. Statistical analysis was performed using ImageJ software.

[0052] Detection indicators: Detection of the expression of oligodendrocyte marker O4+ and myelin MBP.

[0053] The expression detection results of the oligodendrocyte marker O4+ are as follows: Figure 6 As shown: the proportion of O4+ cells in the 40μM and 80μM ginsenoside Rg2 groups was 65.3±5.2, significantly higher than that in the model control group (32.5±4.8, P<0.01); the expression results of myelin protein MBP are as follows. Figure 7 As shown, MBP positive expression was significantly increased (1.58±0.07), and the formation of myelin-like structures was increased, which was significantly higher than that in the model group (1.00±0.0, P<0.01), indicating that ginsenoside Rg2 can promote OPC differentiation and myelin regeneration.

[0054] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of a ginsenoside Rg2 in the preparation of drugs for the treatment or prevention of multiple sclerosis.

2. The application of ginsenoside Rg2 in the preparation of a drug for inhibiting inflammation of the central nervous system, characterized in that, The ginsenoside Rg2 is used to reduce the expression level of pro-inflammatory factors, while promoting the differentiation of regulatory T cells and inhibiting the infiltration of immune cells into the central nervous system.

3. The application of ginsenoside Rg2 in the preparation of neuroprotective drugs, characterized in that, The ginsenoside Rg2 is used to inhibit the activation of the NLRP3 signaling pathway and reduce neuronal pyroptosis.

4. The application of a ginsenoside Rg2 in the preparation of drugs that promote myelin regeneration.

5. The application according to claim 4, characterized in that, The ginsenoside Rg2 is used to prevent the immune system from destroying myelin and reduce the ratio of myelin loss area to white matter area.

6. The application according to claim 4, characterized in that, The ginsenoside Rg2 is used to upregulate the expression of oligodendrocyte transcription factors, promote the proliferation and differentiation of oligodendrocyte precursor cells, and promote myelin regeneration.

7. The application of a ginsenoside Rg2 in the preparation of a drug that inhibits microglia activation.

8. A medicament for treating or preventing multiple sclerosis, comprising a pharmaceutically acceptable carrier, characterized in that, It also includes ginsenoside Rg2.

9. The medicament for treating or preventing multiple sclerosis according to claim 8, characterized in that, The concentration of ginsenoside Rg2 is 20-80 μmol / L.

10. The medicament for treating or preventing multiple sclerosis according to claim 8 or 9, characterized in that, The extraction method of ginsenoside Rg2 includes the following steps: One or more of the roots, stems and leaves of plants in the genus Panax of the Araliaceae family are crushed and extracted by reflux at 80°C with a 70%-80% ethanol aqueous solution to obtain an extract. The above extract was concentrated under reduced pressure until no alcohol odor was detected, and then loaded onto a D101 macroporous resin column. It was eluted sequentially with ethanol aqueous solutions of 30%, 50%, and 70% (v / v), and the eluent of the 70% ethanol aqueous solution was collected. The above eluent was purified by high performance liquid chromatography (HPLC) using a C18 column, a mobile phase of acetonitrile-water = 25:75, a flow rate of 1.0 mL / min, and a detection wavelength of 203 nm. The chromatographic peaks with retention times of 12.5–13.5 min were collected and then freeze-dried to obtain ginsenoside Rg2.

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