Application of tenuifolin in preparation of medicine for treating multiple sclerosis

Abstract

The invention discloses application of tenuifolin in preparation of a medicine for treating multiple sclerosis, and belongs to the technical field of biological pharmacy. Aiming at the problem that the treatment effect and the specific action mechanism of TEN on the multiple sclerosis disease are not clear yet, an EAE model group of a mouse is used for simulating the multiple sclerosis disease, meanwhile, a TEN intervention group is set for simulating the multiple sclerosis disease and then TEN intervention is carried out, and it can be found through experimental comparison that compared with mice of the EAE model group, the mice of the TEN intervention group are more obvious in effect. The average neurological function score is obviously reduced, the clinical symptoms and the demyelination degree are alleviated, the MBP expression is increased, and the inflammatory infiltration is reduced; in addition, TEN intervention can significantly reduce the increase of TLR4 / NF-kappa B protein in mouse spinal cord tissues of an EAE model group. The TEN can inhibit activation of a TLR4 / NF-kappa B pathway to effectively relieve demyelination and abnormal behaviors of mice of an EAE model group and slow down inflammatory response, so that the TEN plays a role in treating the mice of the EAE model group.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutics, and particularly to the application of tenuifolin in the preparation of a drug for treating multiple sclerosis. Background Art

[0002] Multiple sclerosis (MS) is a chronic autoimmune, inflammatory demyelinating central nervous system (CNS) disease characterized by immune abnormalities. Its pathogenesis mainly involves immune system abnormalities, which lead to the invasion of immune cells into the CNS, triggering demyelination and secondary axonal degeneration. MS usually occurs between the ages of 20 and 40 and is one of the most common causes of neurological dysfunction in young people. Its symptoms are diverse, including sensory disturbances, visual problems, movement and coordination difficulties, spasticity, fatigue, pain, and cognitive decline. Although the exact pathological mechanism of MS is not fully understood, it is known that dysregulated immune responses play a central role in CNS damage in MS. Currently, immunosuppressive or immunomodulatory strategies are mainly used clinically to slow down the disease progression and reduce the relapse rate. CD4 + T cells can be divided into multiple functionally different subsets, including Th1, Th2, Th17, and regulatory T cells (Tregs). These CD4 + T cells play a key role in the pathogenesis of MS. Studies have found that a large number of CD4 + T cell infiltrations can be observed in the CNS of MS patients. These cells and the cytokines they release attack neurons and myelin sheaths, triggering inflammation and nerve damage. Therefore, they are considered key cells and mediators of the autoimmune response in MS. At the same time, the imbalance between different CD4 + T cell subsets plays a crucial role in the pathogenesis of the experimental autoimmune encephalomyelitis (EAE) model.

[0003] Tenuifolin (TEN) belongs to the main characteristic component of the traditional Chinese medicine Polygala tenuifolia Willd. and has a wide range of pharmacological activities, such as neuroprotection, anti-inflammatory, antidepressant, and anti-aging effects. The chemical structure is as shown Figure 1 . A large number of studies have confirmed that TEN plays a key role in neuroprotection. It can reduce the damage caused by Aβ by regulating autophagy. 25-35Inflammatory response in an induced Alzheimer's disease cell model. Meanwhile, TEN can effectively protect SH-SY5Y cells from damage caused by Aβ42 oligomer-induced microglia-mediated inflammation and oxidative stress by inhibiting the activation of the NF-κB signaling pathway. In addition, in a mouse model of Parkinson's disease induced by MPTP, TEN effectively improved the degeneration of dopaminergic neurons through its antioxidant and anti-inflammatory properties. However, there is currently no research on the therapeutic effect and specific mechanism of action of TEN on multiple sclerosis by simulating multiple sclerosis disease through an EAE mouse model. Summary of the Invention

[0004] In view of the above problems, the present invention aims to provide the application of tenuifolisaponin in the preparation of a drug for the treatment of multiple sclerosis, and to study the therapeutic effect and specific mechanism of action of TEN on multiple sclerosis by simulating multiple sclerosis disease through an EAE model of mice.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides the application of tenuifolisaponin in the preparation of a drug for the treatment of multiple sclerosis.

[0006] Specifically, tenuifolisaponin plays a role in the treatment of multiple sclerosis by inhibiting the activation of the TLR4 / NF-κB signaling pathway.

[0007] Specifically, tenuifolisaponin inhibits the activation of the TLR4 / NF-κB signaling pathway by downregulating the proportion of pro-inflammatory Th1 / Th17 cells and upregulating the proportion of Tregs anti-inflammatory cells.

[0008] On the other hand, the present invention also provides the application of tenuifolisaponin in the preparation of a TLR4 / NF-κB signaling pathway inhibitor.

[0009] On the other hand, the present invention also provides the application of the TLR4 / NF-κB signaling pathway inhibitor as described above in the preparation of a drug for the treatment of multiple sclerosis.

[0010] The beneficial effects of the present invention are: In this invention, mice were randomly divided into a normal group, an EAE model group, and a TEN intervention group. The EAE model group was used to simulate multiple sclerosis, and the TEN intervention group was used to simulate the TEN intervention after multiple sclerosis. Through experimental comparison, it was found that compared with the mice in the EAE model group, the average neurological function score of the mice in the TEN intervention group was significantly reduced, the clinical symptoms and the degree of myelin loss were alleviated, the MBP expression increased, and the inflammatory infiltration decreased. These results all indicate that TEN can effectively relieve the myelin loss and behavioral abnormalities of the mice in the EAE model group and has an obvious therapeutic effect on multiple sclerosis. Further, by studying the expression of TLR4 / NF-κB signaling pathway proteins, it was found that compared with the normal group, the expression of TLR4 / NF-κB proteins in the spinal cord tissue of the mice in the EAE model group was significantly increased, and after TEN intervention, its expression level was significantly reduced. This indicates that TEN may play a therapeutic role in the mice in the EAE model group by inhibiting the activation of the TLR4 / NF-κB pathway and slowing down the inflammatory response. This discovery provides corresponding experimental basis for the application of TEN in the treatment of multiple sclerosis and is expected to apply TEN in the clinical treatment drugs for multiple sclerosis. Description of the Drawings

[0011] Figure 1 It is the chemical structural formula of tenuifolisaponin.

[0012] Figure 2 It is the comparison result of the clinical scores of the mice in each group of this invention.

[0013] Figure 3 It is the MBP expression and spinal cord myelin loss of the mice in each group of this invention.

[0014] Figure 4 It is the result of the inflammatory infiltration and TLR4 / NF-κB pathway change in the spinal cord tissue of the mice in each group of this invention.

[0015] Figure 5 It is the pro-inflammatory IFN-γ in the spleen and lymph nodes of the mice in each group of this invention + CD4 + T cell ratio.

[0016] Figure 6 It is the pro-inflammatory IL-17 in the spleen and lymph nodes of the mice in each group of this invention + CD4 + T cell ratio.

[0017] Figure 7 It is the anti-inflammatory Foxp3 in the spleen and lymph nodes of the mice in each group of this invention + CD4 + T cell ratio.

[0018] Figure 8 Changes in the secretion of inflammatory factors in splenocytes of mice in each group in the present invention. Specific embodiments

[0019] In order to enable those of ordinary skill in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0020] 1. Experimental materials: 1.1 Animals: 8-week-old male C57BL / 6 mice, weighing 18 - 20 g, a total of 30 mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal production license number: SYXK (Jing) 2022 - 0052. They were housed in an SPF-class animal room at a temperature of 20 - 22 °C, humidity of 40% - 50%, and a 12-hour light-dark cycle, with free access to food and water. The experimental protocol was approved by the Animal Experiment Ethics Committee of Shanxi University of Traditional Chinese Medicine (approval number: AWE202404333).

[0021] 1.2 Reagents: MOG 35-55Polypeptide (BioLabo Technology Co., Ltd., batch number: SY0437); Mycobacterium tuberculosis H37Ra (tuberculosis myco-bacterium, TB, BD Bioscience, batch number: 231141); Pertussis toxin (PTX, Sigma, batch number: 516561); Complete Freund's adjuvant (CFA, Sigma, batch number: F5881); TEN (Shanghai Ronghe Pharmaceutical Co., Ltd., batch number: 20183-47-5); Luxol fast blue staining kit (LFB, Solarbio, batch number: G3245); Hematoxylin-eosin staining kit (HE, Solarbio, G1120); Mouse interferon-γ kit (interferon-gamma, IFN-γ, Solarbio, batch number: SEKM-0031); Mouse interleukin 17 kit (interleukin 17, IL-17, Solarbio, batch number: SEKM-0018); Mouse transforming growth factor β kit (transforming growth factor β, TGF-β, Solarbio, batch number: SEKM-0035); Flow antibody IFN-γ (Thermo Fisher Scientific, batch number: 12-7311-82); Flow antibody IL-17 (Thermo Fisher Scientific, batch number: 17-7177-81); Flow antibody forkhead transcription factor 3 (Foxp3, Thermo Fisher Scientific, batch number: 11-5773-82); Toll-like receptor 4 (TLR4, Proteintech, batch number: 19811-1-AP); Myelin basic protein (MBP, Abcam, batch number: ab40390); Myeloid differentiation factor 88 (MyD88, Abclonal, batch number: A0980); Nuclear factor kappa-B antibody (NF-κB, CST, batch number: D14E12); p-NF-κB (CST, batch number: 93H1); β-actin (Bioworld, batch number: AP0060);HRP-labeled goat anti-rabbit secondary antibody (Boster, lot number: BA1055); RIPA lysis buffer (Servicebio, lot number: G2002).;

[0022] 1.3 Instruments: Cryostat (leica, CM 1950); Fluorescence microscope (leica, DM40008 / DFC450C); 4°C centrifuge (thermo fisher scientific, Megafuge 8R); Multifunctional full-wavelength microplate reader (thermo fisherscientific, Varioskan LUX); Flow cytometer (BD bioscience, Accuri C6); Electrophoresis apparatus (bio-rad, 165-8004).

[0023] 2. Experimental methods: 2.1 Experimental grouping and model preparation: The mice were randomly and evenly divided into a normal group (n = 10), an EAE model group (n = 10), and a TEN intervention group (n = 10). The MOG 35-55 polypeptide was diluted to 5 g·L -1 , and after mixing with an equal volume of CFA (containing TB), 100 μL was subcutaneously injected at 4 random points around the spine of each mouse in the EAE model group and the TEN intervention group. On the 0th day and the 2nd day after immunization, PTX (400 ng per mouse per injection) was intraperitoneally injected to enhance the immune response. The normal group was injected with an equal volume of normal saline. Starting from the 0th day of immunization, the mice in the TEN intervention group were gavaged with 30 mg·kg -1 ·d -1 TEN prepared with a 0.5% carboxymethylcellulose sodium (CMC-Na) solution, while the normal group and the EAE model group were gavaged with an equal volume of CMC-Na solution for 3 weeks.

[0024] 2.2 Nerve function scoring criteria: The behavioral changes of the mice in each group were recorded at the same time every day, and the nerve function of the mice was evaluated using a general 5-point scoring method. The specific scoring criteria are shown in Table 1 below.

[0025] Table 1 Nerve function scoring criteria: ; 2.3 Tissue specimen preparation: On the 21st day after immunization, a pentobarbital sodium solution with a concentration of 30 mg·mL -1 was prepared with normal saline, and all 3 groups of mice were administered at 50 mg·kg-1 The mice in each group were anesthetized by intraperitoneal injection of sodium pentobarbital. Under sterile conditions, the spleens and lymph nodes of the mice in each group were removed and placed in 15 mL centrifuge tubes containing cell culture medium. The enzyme digestion solution was injected into the spleen tissue using a syringe, and then the tissue was minced or squeezed to release the cells. The mixture containing spleen cells was filtered through a sieve to remove large cell clumps and tissue fragments. The cell suspension was centrifuged, the supernatant was discarded, red blood cell lysate was added to remove red blood cells, and after centrifuging to remove the supernatant, 1% BSA was added to resuspend the cells to obtain mononuclear cells. The method for preparing mononuclear cells from lymph nodes was the same as the above process.

[0026] After that, 5 mice were randomly selected from each group for cardiac perfusion with normal saline. After complete perfusion, 4% paraformaldehyde was perfused for fixation. The spinal cord tissue was dissected for fixation, dehydration, OCT embedding, and quick-freezing in liquid nitrogen. The spinal cord tissue was made into frozen sections with a thickness of 10 μm and placed at -80 °C for subsequent fast blue, HE, and immunofluorescence staining. After complete cardiac perfusion with normal saline in the remaining 5 mice in each group, the spinal cord tissue was dissected and placed at -80 °C for subsequent Western Blot detection.

[0027] 2.4 Detection of myelin loss in spinal cord tissue by fast blue staining: The spinal cord tissue sections were immersed in 70% ethanol solution for 15 min, then the sections were taken out and placed in the pre-prepared fast blue staining solution, and incubated overnight at 60 °C. Subsequently, the sections were successively placed in 95% ethanol, deionized water, and 0.05% lithium carbonate solution for thorough washing. Then, gradient dehydration with ethanol and transparency treatment with xylene were carried out, and finally, the sections were sealed with neutral resin. The processed sections were placed under a microscope to observe the pathological conditions of the spinal cord tissue and photographs were taken.

[0028] 2.5 Detection of inflammatory infiltration in spinal cord tissue by HE staining: The sections were first placed in hematoxylin staining solution for 5 min, differentiated with the differentiation solution for 1 min after removing the floating color, washed twice with deionized water (3 min each time), then placed in eosin staining solution for 1 min, successively immersed in 75%, 85%, 95% ethanol, and absolute ethanol for dehydration for 3 s respectively, then immersed in absolute ethanol for 1 min, transparentized with xylene, and sealed with neutral resin. Finally, the inflammatory infiltration in the spinal cord tissue was observed and detected under the microscope.

[0029] 2.6 Detection of MBP expression in spinal cord tissue by immunofluorescence: After the frozen sections were restored to room temperature, the embedding agent was washed away with 1×PBS, 1% BSA + 0.3% Triton X-100 was added for blocking for 30 min, and the diluted primary antibody against MBP was added and incubated overnight in a 4 °C refrigerator. The next day, it was washed three times with 1×PBS, the fluorescent secondary antibody was added, incubated at room temperature for 2 hours, then washed three times with 1×PBS again, and after sealing the slices, the myelin loss was observed under the microscope.

[0030] 2.7 Western blot detection of the protein expression levels of MBP, TLR4, NF-κB, and p-NF-κB in spinal cord tissues: Mouse spinal cord tissues were taken, and proteins were extracted using RIPA lysis buffer. The protein concentration was determined by the BCA method. 20 μg of protein was separated by SDS-PAGE, wet-transferred to a PVDF membrane, blocked with 5% skim milk for 1 hour, and primary antibodies against MBP, TLR4, NF-κB, and p-NF-κB were added and incubated overnight at 4°C. The next day, the membrane was washed 3 times with TBST, the corresponding secondary antibody was added, and the incubation was carried out at room temperature for 1 hour. Then, the membrane was washed 4 times with TBST. ECL chemiluminescence was used for color development, and the protein expression was detected by a gel imaging analyzer. The gray value of the band was analyzed using Image J software.

[0031] 2.8 Flow cytometry for detecting T cells: The single-cell suspensions of the spleens and lymph nodes of the EAE model group and TEN intervention group mice obtained in 2.3 were aliquoted into corresponding flow cytometry tubes, and the flow cytometry antibodies CD4, IFN-γ, IL-17, and Foxp3 for detection were added, and they were incubated in the dark at room temperature for 20 min. They were washed 3 times with PBS, and finally 500 μL of PBS was added to each tube, and detection was carried out using a flow cytometer.

[0032] 2.9 ELISA for detecting inflammatory factors in the supernatant of spleen cells: The single nuclear cells of the spleen obtained in 2.3 were cultured in an incubator for 72 h, the cell supernatant was collected, and the contents of IFN-γ, IL-17, and TGF-β in the culture medium were detected according to the kit instructions.

[0033] 2.10 Statistical analysis: The experimental data were statistically analyzed using Graphpad Prism 8.0 software, and the data were expressed as ±s. Image J software was used to analyze the proportion of inflammatory infiltration and the area of myelin loss. After normal distribution and homogeneity of variance tests, one-way ANOVA was used for comparison among multiple groups, and the t-test was used for pairwise comparison between different groups. P <0.05 indicates a significant difference.

[0034] 3. Experimental results: 3.1 Neurological function scores of mice in each group: The comparison results of the neurological function scores of mice in each group are shown in the appendix Figure 2 as follows. It can be seen from the appendix Figure 2 that the mice in the EAE model group began to show neurological dysfunction on the 11th day, with manifestations such as decreased tail tension and gait disorders, while the clinical scores of EAE in the TEN intervention group mice at each time period were lower than those in the EAE model group ( P <0.05).

[0035] 3.2 Demyelination condition: The MBP expression and spinal cord demyelination conditions of mice in each group are shown in the appendix Figure 3 as follows. Among them, A and C show the MBP expression of mice in each group, and B shows the spinal cord demyelination condition. As can be seen from the appendix Figure 3 , compared with the normal group, mice in the EAE model group showed extensive spinal cord demyelination ( P <0.01 or P <0.001), and the expression of the myelin marker protein MBP was significantly decreased ( P <0.001); compared with the EAE model group, the demyelination range of mice in the TEN intervention group was significantly reduced ( P <0.01 or P <0.001), and the MBP expression was significantly increased ( P <0.01).

[0036] 3.3 Inflammatory microenvironment condition of spinal cord tissue: The inflammatory infiltration and changes in the TLR4 / NF-κB pathway in the spinal cord tissue of mice in each group are shown in the appendix Figure 4 as follows. Among them, A shows the HE staining results (scale bar = 70 µm) and statistical results of inflammatory infiltration in the spinal cord tissue of mice in each group, and B shows the protein expression levels of TLR4, NF-κB, and p-NF-κB in each group of mice. As can be seen from the appendix Figure 4 , compared with the normal group, extensive inflammatory infiltration was visible in the EAE model group ( P <0.001); compared with the EAE model group, the infiltration of inflammatory cells in the TEN intervention group was significantly reduced ( P <0.001); compared with the normal group, the expression levels of TLR4 and p-NF-κB proteins in the spinal cord tissue of mice in the EAE model group were both significantly increased ( P <0.001); compared with the EAE model group, the expression levels of these proteins in the TEN intervention group were significantly decreased ( P <0.01 or P <0.001).

[0037] 3.4 Differentiation of T cells in spleen and lymph nodes: The comparison results of T cell subsets in the spleen and lymph nodes of mice in the EAE model group and the TEN intervention group are shown in the appendix Figures 5 - 7 as follows. Among them, Figure 5 shows the proportion of pro-inflammatory IFN-γ + CD4 + T cells in the spleen and lymph nodes of each group of mice, Figure 6 shows the proportion of pro-inflammatory IL-17 + CD4 + T cells in the spleen and lymph nodes of each group of mice,Figure 7 The proportion of anti-inflammatory Foxp3 + CD4 + T cells in the spleen and lymph nodes of each group of mice. As can be seen from the appendix Figure 5 Among them, compared with the EAE model group, the number of pro-inflammatory IFN-γ + CD4 + T cells in the spleen and lymph node tissues of the TEN intervention group of mice was significantly decreased ( P <0.001 or P <0.01). As can be seen from Figure 6 Among them, compared with the EAE model group, the number of pro-inflammatory IL-17 + CD4 + T cells in the spleen and lymph node tissues of the TEN intervention group of mice was significantly decreased ( P <0.01 or P <0.001). As can be seen from Figure 7 Among them, compared with the EAE model group, the anti-inflammatory Foxp3 + CD4 + T cells in the spleen and lymph nodes of the TEN intervention group of mice were significantly increased ( P <0.01 or P <0.01).

[0038] 3.5 Secretion of inflammatory factors in the supernatant of splenocytes: The changes in the secretion of inflammatory factors by mouse splenocytes are shown in the appendix Figure 8 As shown. Among them, A is the expression of the pro-inflammatory factor IFN-γ, B is the expression of the pro-inflammatory factor IL-17, and C is the expression of the anti-inflammatory factor TGF-β. As can be seen from the appendix Figure 8 Among them, compared with the normal group, the contents of the pro-inflammatory factors IFN-γ, IL-17 and the anti-inflammatory factor TGF-β in the culture medium of splenocytes of the EAE model group of mice were significantly increased ( P <0.05 or P <0.01); after TEN intervention, the contents of the inflammatory factors IFN-γ, IL-17 in the culture medium of mouse splenocytes were significantly decreased ( P <0.05 or P <0.01), and the content of the anti-inflammatory factor TGF-β was further increased ( P <0.01).

[0039] From the above experimental results, it can be seen that this study successfully established an EAE model in mice. After TEN intervention, the average neurological function score of the mice decreased significantly, and the clinical symptoms and the degree of demyelination were alleviated. The expression of MBP increased, and the inflammatory infiltration decreased. These results indicate that TEN can effectively relieve demyelination and behavioral abnormalities in mice in the EAE model group and has an obvious therapeutic effect. The TLR4 / NF-κB signaling pathway is a key pathway for inflammatory signal transduction and is often closely related to cell differentiation, proliferation, apoptosis, and inflammatory responses. Therefore, inhibiting the TLR4 / NF-κB signaling pathway may be an important way to treat MS / EAE. This study also found that compared with the normal group, the expression of TLR4 / NF-κB proteins in the spinal cord tissue of mice in the EAE model group was significantly increased, while after TEN intervention, its expression level was significantly decreased. This indicates that TEN may play a therapeutic role in mice in the EAE model group by inhibiting the activation of the TLR4 / NF-κB pathway and slowing down the inflammatory response.

[0040] The occurrence of CNS inflammation requires the activation of T cells in the periphery and their entry into the CNS, where they are re-activated by antigen-presenting cells presenting self-antigens. This re-activation of T cells triggers various cells to produce soluble mediators, which in turn recruit other inflammatory cells. CD4 + T cells play a key role in MS. In the initial stage of MS, CD4 + T cells cross the blood-brain barrier and migrate towards the center, activating immune cells and releasing inflammatory factors, leading to demyelination of the spinal cord white matter. Activated peripheral naive CD4 + T cells can differentiate into regulatory T cells such as Th1, Th2, Th17, and Treg, which participate in different types of immune responses. Th1 is a CD4 + T cell subset that produces pro-inflammatory cytokines such as IFN-γ, while Th17 produces pro-inflammatory cytokines such as IL-17. Treg cells are another special type of CD4 + T cells that can secrete the anti-inflammatory factor TGF-β and maintain the balance of the immune system and autoimmune tolerance by inhibiting the activity of other immune cells, especially inhibiting autoimmune reactions, to prevent the occurrence of excessive inflammation and autoimmune diseases. The key transcription factor Foxp3 of Treg cells is crucial for its development and function and is considered a marker of Treg cells. This study showed that compared with the EAE model group, the number of pro-inflammatory IL-17 + CD4 + T cells and IFN-γ + CD4 + T cells in the spleen and lymph node tissues of EAE mice intervened with TEN decreased significantly, while the anti-inflammatory Foxp3 + CD4 +The number of T cells increased significantly. This indicates that TEN can regulate peripheral CD4 + T cell subsets, that is, by downregulating the proportion of pro-inflammatory Th1 / Th17 cells and upregulating the proportion of Tregs anti-inflammatory cells, to reduce the central inflammatory response, demyelination and clinical symptoms of EAE mice.

[0041] In summary, TEN can significantly improve demyelination and inflammatory responses in EAE mice. Its mechanism of action may be related to inhibiting the activation of the TLR4 / NF-κB signaling pathway and maintaining the homeostasis of CNS immune cells. This finding provides corresponding experimental evidence for the application of TEN in the treatment of multiple sclerosis.

[0042] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. Use of tenuifolisaponin in the preparation of a drug for treating multiple sclerosis.

2. The use according to claim 1, characterized in that: tenuifolisaponin plays a role in the treatment of multiple sclerosis by inhibiting the activation of TLR4 / NF-κB signaling pathway.

3. The use according to claim 2, characterized in that: tenuifolisaponin inhibits the activation of TLR4 / NF-κB signaling pathway by downregulating the proportion of pro-inflammatory Th1 / Th17 cells and upregulating the proportion of Tregs anti-inflammatory cells.

4. Use of tenuifolisaponin in the preparation of a TLR4 / NF-κB signaling pathway inhibitor.

5. Use of the TLR4 / NF-κB signaling pathway inhibitor according to claim 4 in the preparation of a drug for treating multiple sclerosis.

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