Application of morusin in preparation of medicine for treating intervertebral disc degeneration

Abstract

The invention discloses application of morusin in preparation of a medicine for treating intervertebral disc degeneration, and the inventor proves that morusin can effectively prevent intervertebral disc degeneration and has a repairing effect on degenerated intervertebral disc tissues through experimental research. Compared with the existing conservative treatment scheme that only clinical symptoms are relieved, the progress of IVDD cannot be prevented, or recurrence may occur in an operative treatment strategy and occurrence of spinal degeneration of adjacent segments is accelerated, the morusin can slow down the progress of IVDD in a body and fundamentally prevent occurrence of IVDD, and therefore the morusin has important significance in prevention and treatment of intervertebral disc degeneration.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of sansin in the preparation of a drug for treating intervertebral disc degeneration. Background Art

[0002] Intervertebral disc degeneration (IVDD) is an important pathological factor leading to neck, shoulder and back pain. The Lancet pointed out in a review of 165 studies that low back pain is the leading cause of disability worldwide. IVDD not only causes great damage to patients physically and mentally, but also imposes a significant economic burden on society.

[0003] The treatment of disc degeneration mainly includes conservative treatment and surgical treatment. Conservative treatment mainly includes drug treatment and non-drug treatment. At present, the drug treatment of disc degeneration mainly relieves pain through drugs, such as non-steroidal anti-inflammatory drugs (NSAIDs), anticonvulsants and / or antidepressants (i.e. gabapentin, pregabalin and duloxetine), patch analgesia, etc. Non-drug treatment is mainly related to rehabilitation treatment, and other treatment measures include peripheral nerve block, epidural injection, facet joint injection and radiofrequency ablation. However, these treatment strategies cannot fundamentally block the progression of the disease. Surgical treatment mainly removes the patient's protruding disc tissue to relieve nerve compression, which can quickly relieve the patient's symptoms, but the side effect of accelerating the degeneration of adjacent segments is difficult to avoid. Therefore, exploring treatment methods that fundamentally prevent the progression of IVDD has received more and more attention.

[0004] Intervertebral disc degeneration (IVDD) is a common inflammatory-related disease. Nucleus pulposus cells are the main functional cells in the intervertebral disc. The inflammatory response of nucleus pulposus cells mediated by NF-κB signaling plays an important role in the occurrence and development of IVDD. Therefore, exploring small molecule drugs that can improve the inflammatory response of nucleus pulposus cells is the core concept of treating IVDD.

[0005] Sancin is a small molecule natural product with a molecular formula of C 25 H 24 O 6 Mulberry is a devinylated flavonoid isolated from the root bark of mulberry. The drug is easy to store and has high stability. Previous literature reports have shown that mulberry has an anti-inflammatory effect in osteoarthritis + diseases, but the in-depth anti-inflammatory mechanism is not clear, and importantly, its role in intervertebral disc degeneration has not been studied. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides the use of mulberry in the preparation of drugs for the treatment of intervertebral disc degeneration. The inventor's experimental research has confirmed that mulberry can effectively prevent intervertebral disc degeneration and has a repairing effect on degenerated intervertebral disc tissue. In in vitro studies, mulberry can inhibit TNF-α-mediated nucleus pulposus inflammatory response, further reduce the production of inflammatory factors induced by TNF-α (such as IL-1β, IL-6, COX-2 and other inflammatory factors), and weaken the inflammatory response of nucleus pulposus cells by inhibiting the production of these inflammatory factors. At the same time, it can reduce the production of matrix decomposition enzymes (such as MMP-13, ADAMTs4, etc.), and can reverse the inhibition of TNF-α-mediated anabolic genes (ACAN, Col-2). The above studies show that mulberry can prevent the destructive effect of the destructive enzyme on intervertebral disc tissue and maintain the integrity of intervertebral disc tissue. In vivo studies, the inventor used rats to acupuncture the coccygeal disc to construct an injury model. After treatment with mulberry, the inventor found that the intervertebral disc degeneration of rats was significantly alleviated. Further mechanism studies have found that when preventing and treating intervertebral disc degeneration, Sansin does not bind to TNFR receptors, nor does it affect the MAPK inflammatory pathway. Instead, it directly enters the nucleus pulposus cells and binds to RELA (p65) in the cells, stabilizing the IKα-P65-P50 complex, inhibiting the phosphorylation of p65 and IKα, and blocking the nuclear translocation of the p65-p50 complex. It then exerts an anti-inflammatory effect by affecting the phosphorylation of NF-κB, ultimately inhibiting the occurrence and development of IVDD.

[0007] Based on the above research, the present invention provides the following technical solutions:

[0008] As a first aspect of the present invention, it provides the use of sansin in preparing a drug for treating intervertebral disc degeneration.

[0009] As a second aspect of the present invention, it provides the use of sansin in preparing a drug for preventing intervertebral disc degeneration.

[0010] As a third aspect of the present invention, a drug for preventing and / or treating intervertebral disc degeneration is provided, wherein the drug contains sansin.

[0011] The drug further includes pharmaceutically acceptable excipients, which include any one or more of diluents, glidants, fillers, emulsifiers, disintegrants, decomposing agents, antioxidants, preservatives, solubilizers, and excipients.

[0012] In an embodiment of the present invention, the medicine is an injection.

[0013] Furthermore, when the drug is an injection, in addition to the diluent (water for injection), it also includes an injection additive for increasing the effectiveness, safety and stability of the injection.

[0014] The study of the embodiments of the present invention also found that sangxinsu has the characteristics of good safety of intraperitoneal injection. Furthermore, the injection is administered by intraperitoneal injection.

[0015] The injection concentration is 5μM~10μM.

[0016] The dosage is 20 mg / kg.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. Compared with the existing conservative treatment schemes that only relieve clinical symptoms but cannot prevent the progression of IVDD, or the surgical treatment strategies that may recur and accelerate the occurrence of adjacent segmental spinal degeneration, the research of the present invention shows that sansin can slow down the progression of IVDD in the body and fundamentally prevent the occurrence of IVDD. Based on this, the use of sansin provided by the present invention in the preparation of drugs for treating intervertebral disc degeneration is of great significance in the prevention and treatment of intervertebral disc degeneration.

[0019] 2. The research in the examples of the present invention also found that sansin has the characteristics of good safety for intraperitoneal injection, which provides a new treatment plan for the prevention and treatment of intervertebral disc degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 This is a side view of the cytotoxicity of sangsin to human NP cells in Example 1;

[0022] Figure 2 The results of AnnexinV / PI detection of cell apoptosis in Example 1;

[0023] Figure 3 The apoptosis rate of each group of human NP cells cultured with different concentrations of sangsin in Example 1;

[0024] Figure 4 HE staining of viscera under different concentrations of sanshinone in Example 1;

[0025] Figure 5 HE staining of the caudal vertebrae sections of rats in the sham operation group, acupuncture injury group, and drug treatment group (drug concentration: 20 mg / kg) in Example 2 after 8 weeks;

[0026] Figure 6 for Figure 5 Statistical graph of histological scores of different treatment groups;

[0027] Figure 7 This is a safranin fast green staining image of the caudal vertebrae sections of rats in the sham operation group, acupuncture injury group, and drug treatment group (drug concentration: 20 mg / kg) after 8 weeks in Example 2;

[0028] Figure 8 for Figure 7 Statistical graph of histological scores of different treatment groups;

[0029] Fig. 9 The immunohistochemical staining results of Aggrecan, iNOS and MMP13 in the caudal vertebrae sections of rats in the sham operation group, acupuncture injury group and drug treatment group (drug concentration: 20 mg / kg) in Example 2 after 8 weeks;

[0030] Fig.10 for Fig. 9 Quantitative analysis results of Aggrecan, iNOS, and MMP13 expression in different treatment groups;

[0031] Fig.11 Statistical analysis of PCR detection of IL-1β, iNOS, and COX2 mRNA expression in rat nucleus pulposus cells in the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration 10 μM) in Example 3;

[0032] Fig.12 Statistical analysis of PCR detection of ADAMTs4 and MMP13 mRNA expression in rat nucleus pulposus cells in the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration 10 μM) in Example 3;

[0033] Fig.13 The results of Western blot detection and quantitative statistical analysis of the expression of iNOS, COX2, ADAMTs4, and MMP13 proteins in the rat nucleus pulposus cells of the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration: 10 μM) in Example 3;

[0034] Fig.14 Statistical analysis of ELISA experimental detection of IL-1β and IL-6 expression in rat nucleus pulposus cells in the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration: 10 μM) in Example 3;

[0035] Fig.15The immunofluorescence staining results of COX2 and MMP13 expression in the nucleus pulposus cells of rats in the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration: 10 μM) in Example 3;

[0036] Fig.16 for Fig.15 Quantitative analysis statistical graphs of different treatment groups;

[0037] Fig.17 The immunohistochemical staining results of COX2 and MMP13 expression in human nucleus pulposus tissue of the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration: 10 μM) in Example 3;

[0038] Fig.18 for Fig.17 Quantitative analysis statistical graphs of different treatment groups;

[0039] Fig.19 Statistical analysis of PCR detection of Aggrecan, COL2, and SOX9 expression in rat nucleus pulposus cells in the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration 10 μM) in Example 4;

[0040] Fig. 20 The results of Western blot detection and quantitative statistical analysis of the expression of Aggrecan, COL2, and SOX9 proteins in the rat nucleus pulposus cells in the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration: 10 μM) in Example 4;

[0041] Fig.21 The immunofluorescence staining results of Aggrecan expression in rat nucleus pulposus cells of the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration: 10 μM) in Example 4;

[0042] Fig. 22 for Fig.21 Quantitative analysis statistical graphs of different treatment groups;

[0043] Fig.23The immunohistochemical staining results of Aggrecan expression in human nucleus pulposus tissue of the control group, TNF-α stimulation group, TNF-α+Morusin (drug concentration: 5 μM), and TNF-α+Morusin (drug concentration: 10 μM) in Example 4;

[0044] Fig.24 for Fig.23 Quantitative analysis statistical graphs of different treatment groups;

[0045] Fig.25 The results of Western blot detection and quantitative statistical analysis of the expression of p-IκBα, IκBα, p-P65 and P65 proteins in the nucleus pulposus cells of different groups in Example 5 after being treated with TNF-α (10 ng / mL) for different time periods (15, 30, 60 minutes) in the absence or presence of Morusin;

[0046] Fig.26 The results of western blot detection and quantitative statistical analysis of the protein expression of P65 and GAPDH in the cytoplasm and P65 and LaminB1 in the nucleus were obtained by treating the rat nucleus pulposus cells with TNF-α (10 ng / mL) for different time periods (0, 15, 30 minutes) in the absence or presence of Morusin in different groups of nucleus pulposus cells in Example 5 and extracting cytoplasmic and nuclear proteins;

[0047] Fig. 27 The immunofluorescence staining results of P65 expression in the nucleus pulposus cells of rats in the control group, TNF-α stimulation group, and TNF-α+Morusin (drug concentration: 10 μM) in Example 5;

[0048] Fig.28 The results of the drug affinity target stability (DARTS) test (DARTS) of human nucleus pulposus cells treated with or without sangsin (10 μM) for 48 h with or without TNF-α (10 ng / mL) intervention in Example 6, and the results of Coomassie brilliant blue staining after protein separation by SDS-PAGE, and the mass spectra of the bands with molecular weights of approximately 200 kDa and 65 kDa analyzed by mass spectrometry;

[0049] Fig.29 The results of Western blotting in Example 6 are obtained after digestion of cell lysates with different concentrations of proteases in the presence or absence of sansin incubation;

[0050] Fig.30 The results of Western blotting in Example 6 are obtained after digestion of cell lysates with different concentrations of proteases in the presence or absence of sansin incubation;

[0051] Fig.31The cell lysate was subjected to a cellular thermal shift assay (CETSA) in Example 6, and the changes in the thermal denaturation temperature of RELA (p65) at different temperatures (55, 60, 65, 70, 75, 80, 85°C) were observed with or without incubation with sansin;

[0052] Fig.32 The CETSA experiment in Example 6 was carried out at 60°C using different concentrations (100, 10, 1, 0.1, 0.01, 0.001 μM) of sansin to observe the changes in the degree of denaturation of RELA (p65);

[0053] Fig.33 A schematic diagram of the optimized three-dimensional structure model of sansin and human p65 protein and an overview of the binding mode in Example 6;

[0054] Fig.34 The 29 docking configurations obtained by docking sxs with human p65 protein by molecular docking in Example 6 are ranked according to their affinities;

[0055] Fig.35 The knockdown effect of siRNA on RELA (P65) in human nucleus pulposus cells was detected by Western blot analysis in Example 6;

[0056] Fig.36 The expression levels of iNOS, COX2, ADAMTs4, and MMP13 mRNA were detected by qRT-PCR in human nucleus pulposus cells transfected with NC and RELA (P65) siRNA in Example 7 in the absence or presence of sansin (10 μM) and without or with TNF-α (10 ng / mL) stimulation for 24 h;

[0057] Fig.37 The expression levels of COL2 and Aggrecan mRNA were detected by qRT-PCR in human nucleus pulposus cells transfected with NC and RELA (P65) siRNA in Example 7 in the absence or presence of sansin (10 μM) and without or with TNF-α (10 ng / mL) stimulation for 24 h;

[0058] Fig.38 The protein expression levels of iNOS, COX2, ADAMTs4, and MMP13 were detected by Western blot analysis in the absence or presence of sangrin (10 μM) and in the absence or presence of TNF-α (10 ng / mL) in human nucleus pulposus cells transfected with NC and RELA (P65) siRNA in Example 7 after 48 h of stimulation;

[0059] Fig.39 for Fig.38Statistics for quantitative analysis of Western blot analysis results;

[0060] Fig.40 The protein expression level of Aggrecan was detected by immunofluorescence technique in human nucleus pulposus cells transfected with NC and RELA (P65) siRNA in Example 7 in the absence or presence of sansin (10 μM) and without or with TNF-α (10 ng / mL) stimulation for 48 h;

[0061] Fig.41 for Fig.40 Quantitative analysis and statistics of immunofluorescence results. DETAILED DESCRIPTION

[0062] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0063] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0064] Morusin: It is a devinylated flavonoid isolated from Morus alba Linn. (mulberry tree) and has various biological activities such as anti-tumor, antioxidant, and antibacterial.

[0065] RELA: RELA is also known as p65 protein. It is one of the important components of nuclear factor-κB and a key protein for activating the NFκB pathway.

[0066] Aggrecan, encoded by the Aggrecan Core Protein (ACAN) gene, is an important component of the extracellular matrix of cartilage cells and plays a key role in the morphogenesis of cartilage and bone.

[0067] Intervertebral disc degeneration: Intervertebral disc degeneration (IVDD) is a pathological change, which is the aging process and damage of the intervertebral disc (IVD). It is caused by a series of complex molecular mechanisms, which eventually destroys the original structure of the intervertebral disc, resulting in a decline in the function of the intervertebral disc to buffer mechanical pressure, causing clinical symptoms such as low back pain.

[0068] Example 1 Sangxinsu has good drug safety

[0069] (1) Different concentrations of sangsin (0, 10 μM, 20 μM, 50 μM) were inoculated into 96-well plates and cultured for 24 hours, 48 ​​hours, and 72 hours, respectively. The cytotoxicity of sangsin on human NP cells was evaluated using the CCK8 detection kit. Within the tested concentration range, sangsin had no significant effect on cell activity ( Figure 1 ).

[0070] (2) Human NP cells were cultured with different concentrations of sangsin (0, 10, 20 μM). After 24 hours of culture, cell apoptosis was measured by flow cytometry. The results showed that there was no significant difference in cell apoptosis among the groups ( Figure 2-3 ).

[0071] (3) Eighteen healthy male rats aged 8-12 weeks were selected and randomly divided into different drug concentration groups (0, 10 mg / kg, 20 mg / kg). Sangsin was dissolved in DMSO and diluted to 2 mg / mL with DMEM / F-12 medium. Different doses (10, 20 mg / kg) were intraperitoneally injected daily according to different subgroups. The control group rats were intraperitoneally injected with an equal amount of DMEM / F-12 / DMSO mixture every day. The injection lasted for 8 weeks. After euthanasia, the internal organs (heart, liver, lung, and kidney) were extracted for HE staining to evaluate the toxicity of different concentrations of Sangsin. No obvious visceral damage was found in the rats ( Figure 4 ).

[0072] Example 2: Sansin can inhibit intervertebral disc degeneration in the tail injury rat model

[0073] A rat intervertebral disc degeneration model was established, and 18 healthy male rats were randomly divided into a sham operation group, acupuncture injury group, and sansin treatment group. After the rats were anesthetized, the skin surface was disinfected with 75% alcohol.

[0074] In the sham operation group, a 20-gauge needle was used to puncture the skin of the 8th and 9th coccygeal vertebrae of the rats parallel to the vertebral body, and then the needle was rotated 360 degrees.

[0075] In the needling group, a 20-gauge needle parallel to the coccyx was inserted into the area between the 8th and 9th coccygeal vertebrae (Co8-Co9) of the rats to a depth of 5 mm. The needle was then rotated 360 degrees. Rats that underwent needling were intraperitoneally injected with a DMEM / F12 / DMSO mixture every day.

[0076] In the needling+morusin group, rats subjected to needling injury were intraperitoneally injected with morusin (drug dose: 20 mg / kg) dissolved in a mixture of DMEM / F12 / DMSO equal to that of the needling injury group every day for 8 weeks. The rats were euthanized and their caudal vertebrae were extracted and histologically examined to evaluate the extent of IVDD in rats in different groups.

[0077] The results are as follows Figure 5-8 As shown in the data, compared with the Needling group, the intervertebral disc height of the Needling+Morusin group was restored, the boundary between the nucleus pulposus and the annulus fibrosus was clearer, the cartilage component was significantly increased, and the number of nucleus pulposus cells was significantly increased, indicating that the acupuncture injury of the rat intervertebral disc had recovered to a certain extent. This shows that Morusin has a therapeutic effect on intervertebral disc degeneration induced by acupuncture injury in rats.

[0078] Immunohistochemical staining was used to detect the expression of iNOS, MMP-13 and ACAN proteins in the rat intervertebral disc injury model. Figure 9-10 As shown in the data, the expressions of inflammatory factor iNOS and matrix destructive enzyme MMP-13 were reduced, and the expression of extracellular matrix component ACAN was increased in the sangsin group, indicating that sangsin has a protective effect on acupuncture injury-induced intervertebral disc degeneration.

[0079] Example 3: Sancin can inhibit inflammation in nucleus pulposus cells in TNF-α-mediated intervertebral disc degeneration

[0080] (1) Rat nucleus pulposus cells were treated with or without sansin (10 μM) in the presence or absence of TNF-α (10 ng / mL). Total RNA was extracted 24 h later, and PCR was used to detect the expression of IL-1β, iNOS, COX2, ADAMTs4, and MMP13 mRNA in nucleus pulposus cells. Figure 11-12 As shown, in the nucleus pulposus cells of rats treated with sangsin (10 μM), the expressions of IL-1β, iNOS, COX2, ADAMTs4, and MMP13 mRNA in the nucleus pulposus cells were decreased, indicating that sangsin inhibited the inflammation in the nucleus pulposus cells in TNF-α-mediated intervertebral disc degeneration.

[0081] (2) Rat nucleus pulposus cells were treated with or without sansin (10 μM) in the presence or absence of TNF-α (10 ng / mL). Total protein was extracted 48 h later, and the expression of iNOS, COX2, ADAMTs4, and MMP13 proteins in nucleus pulposus cells was detected by WB. Fig.13As shown, in the rat nucleus pulposus cells treated with sangsin (10 μM), the expression of iNOS, COX2, ADAMTs4, and MMP13 decreased, indicating that sangsin inhibited the inflammation in the nucleus pulposus cells in TNF-α-mediated intervertebral disc degeneration.

[0082] (3) Rat nucleus pulposus cells were treated with or without sansin (10 μM) in the presence or absence of TNF-α (10 ng / mL). After 48 h, the expression of IL-1β and IL-6 in nucleus pulposus cells was detected using the ELASA detection kit. Fig.14 As shown, in the rat nucleus pulposus cells treated with sangsin (10 μM), the expression of intracellular IL-1β and IL-6 decreased, indicating that sangsin inhibited the inflammation in the nucleus pulposus cells in TNF-α-mediated intervertebral disc degeneration.

[0083] (4) Rat nucleus pulposus cells were treated with or without sansin (10 μM) in the presence or absence of TNF-α (10 ng / mL). Immunofluorescence was used to detect the expression of COX2 and MMP13 in nucleus pulposus cells 48 hours later. Figure 15-16 As shown, in the nucleus pulposus cells of rats treated with sangsin (10 μM), the fluorescence intensity of the cells was significantly lower than that of the group stimulated with TNF-α alone, and the expression of COX2 and MMP13 decreased, indicating that sangsin inhibited the inflammation in the nucleus pulposus cells in TNF-α-mediated intervertebral disc degeneration.

[0084] (5) Human nucleus pulposus tissue was treated with or without sangsin (10 μM) for 24 h with or without TNF-α (10 ng / mL) intervention. Immunohistochemical staining was then performed to detect the expression of COX2 and MMP13 in the nucleus pulposus tissue. The control group was treated with no TNF-α intervention and sangsin treatment, and the TNF- group was treated with TNF-α (10 ng / mL, F12 medium) only.

[0085] +Moruisn means human nucleus pulposus tissue was treated with TNF-α (10 ng / mL) and sansin (10 μM) for 24 h. Figure 17-18 As shown, the expression of COX2 and MMP13 in the treatment group was significantly decreased, indicating that sansin inhibited the inflammation in the nucleus pulposus cells in TNF-α-mediated intervertebral disc degeneration.

[0086] Example 4: Sancin can inhibit TNF-α-mediated degradation of extracellular matrix of nucleus pulposus cells

[0087] (1) Rat nucleus pulposus cells were treated with or without sangsin (10 μM) in the presence or absence of TNF-α (10 ng / mL). Total RNA was extracted 24 hours later, and the expression of Aggrecan, Col2, and SOX9 mRNA in nucleus pulposus cells was detected by PCR. The results are shown in Fig.19 As shown, the expression of extracellular matrix Aggrecan, Col2, and SOX9 increased in rat nucleus pulposus cells treated with sangsin (10 μM), indicating that sangsin inhibited TNF-α-mediated degradation of the extracellular matrix.

[0088] (2) Rat nucleus pulposus cells were treated with or without sangsin (10 μM) in the presence or absence of TNF-α (10 ng / mL). Total protein was extracted 72 h later, and the expression of Aggrecan, Col-II, and SOX-9 proteins in nucleus pulposus cells was detected by WB. Fig. 20 As shown, the expression of extracellular matrix Aggrecan, Col2, and SOX9 was increased in rat nucleus pulposus cells treated with sangsin (10 μM), indicating that sangsin inhibited TNF-α-mediated degradation of the extracellular matrix.

[0089] (3) Rat nucleus pulposus cells were treated with or without sangsin (10 μM) in the presence or absence of TNF-α (10 ng / mL). Immunofluorescence was used to detect the expression of Aggrecan in nucleus pulposus cells 72 hours later. Figure 21-22 As shown, in the rat nucleus pulposus cells treated with sangsin (10 μM), the fluorescence intensity in the extracellular matrix was significantly higher than that in the group stimulated by TNF-α alone, and the expression of Aggrecan was increased, indicating that sangsin inhibited the degradation of the extracellular matrix mediated by TNF-α.

[0090] (4) Human nucleus pulposus cells were treated with or without Moruisn (10 μM) for 24 h with or without TNF-α (10 ng / mL) intervention. Then, the human nucleus pulposus cells were subjected to immunohistochemical staining to detect the expression of Aggrecan. The control group was not intervened by TNF-α and was not treated with Moruisn, the TNF- was only intervened with TNF-α (10 ng / mL, F12 culture medium), and the TNF-α+Moruisn group was intervened with TNF-α (10 ng / mL) and Moruisn (10 μM) was used to treat human nucleus pulposus cells for 24 h. The staining results are shown in Figure 23-24 As shown, the expression of Aggrecan in the treatment group was significantly increased, indicating that sansin treatment could inhibit TNF-α-mediated degradation of extracellular matrix in nucleus pulposus cells.

[0091] Example 5: Sancin inhibits TNF-α / NF-κB signaling pathway in nucleus pulposus cells

[0092] (1) Rat nucleus pulposus cells were treated with TNF-α (10 ng / mL) for different time periods (15, 30, and 60 min) in the absence or presence of sanshin. The protein levels of p-IκBα, IκBα, p-P65, and P65 were determined by Western blotting. The protein band density was quantitatively analyzed using ImagJ software. The results are shown in Fig.25 As shown, TNF-α effectively enhanced the phosphorylation of IκBα and P65 in a time-dependent manner, while sansin significantly inhibited TNF-α-induced P65 phosphorylation and IκBα degradation.

[0093] (2) In the absence or presence of sansin, rat nucleus pulposus cells were treated with TNF-α (10 ng / mL) for different time periods (0, 15, 30 min), cytoplasmic and nuclear proteins were extracted, and protein expression of P65 and GAPDH in the cytoplasm and P65 and LaminB1 in the nucleus was determined by Western blotting. Protein band density was quantitatively analyzed using ImagJ software. The results are shown in Fig.26 As shown in the figure, in the presence of TNF-α, the expression of P65 in the cytoplasm decreased, while the expression of P65 in the nucleus increased. The nuclear translocation of P65 was inhibited in nucleus pulposus cells treated with sanshin.

[0094] (3) In the presence or absence of TNF-α (10 ng / mL), rat nucleus pulposus cells were treated with or without sansin (10 μM), and the expression and localization of P65 protein were detected by immunofluorescence staining. Fig. 27 As shown, TNF-α promoted the nuclear translocation of P65 in nucleus pulposus cells, the expression of P65 in the cytoplasm decreased, while the expression of P65 in the nucleus increased, and sansin treatment inhibited the nuclear translocation of P65.

[0095] Example 6: Sancin protects IVDD by inhibiting TNF / NF-κB signaling pathway by binding to RELA protein

[0096] (1) Human nucleus pulposus cells were treated with or without sangsin (10 μM) with or without TNF-α (10 ng / mL) intervention. Drug affinity target stability (DARTS) assay was performed 48 h later, and proteins were separated by SDS-PAGE. Fig.28 As shown in Figure 2, the bands with molecular weights of approximately 200 kDa and 65 kDa were protected by sansin. The bands in this region were analyzed by mass spectrometry. Cell lysates were digested with different concentrations of proteases in the presence or absence of sansin, and the results were shown in Figure 2. Figure 29-30 As shown, sansin was found to protect RELA (p65) from protease digestion under different conditions.

[0097] (2) Cellular thermal shift assay (CETSA) was performed to study the changes in the thermal denaturation temperature of RELA (p65) at different temperatures with or without sansin. Compared with the control group, sansin prevented the denaturation of RELA (p65) at different temperatures, especially at 70°C. Fig.31 Then, CETSA experiments were performed at 60°C using different concentrations of sansin, and the results showed that sansin prevented RELA (p65) denaturation in a dose-dependent manner ( Fig.32 ).

[0098] (3) Molecular docking of sansin and human p65 protein was performed, and the analysis results were as follows Figure 33-34 As shown, a total of 29 docking conformations were obtained by molecular docking, and the highest score according to its affinity ranking was -14.75 kcal / mol. Therefore, the conformation of Rank 1 is the most likely binding mode of Sansinol binding to RELA.

[0099] Example 7 RELA is required for sansin to regulate inflammation in nucleus pulposus cells.

[0100] In order to clarify the role of RELA (P65) in mediating Morusin to alleviate inflammation in human nucleus pulposus cells, the expression of RELA (P65) was knocked down using siRNA ( Fig.35 ).

[0101] (1) Nucleus pulposus cells transfected with NC or RELA (P65) siRNA were treated with or without sansin (10 μM) in the presence or absence of TNF-α (10 ng / mL). Total RNA was extracted 24 h later, and the mRNA levels of inflammatory factors (iNOS and COX2), decomposition markers (ADAMTs4 and MMP13), and anabolic markers (Aggrecan and COL2) were detected by qRT-PCR. The results are shown in Figure 36-37 As shown, in cells where RELA(P65) was downregulated, sansin-mediated inhibition of TNF-α activity was almost completely lost.

[0102] (2) Nucleus pulposus cells transfected with NC or RELA (P65) siRNA were treated with or without sansin (10 μM) in the presence or absence of TNF-α (10 ng / mL). Total protein was extracted 48 h later, and the protein expression levels of inflammatory factors (iNOS and COX2), decomposition markers (ADAMTs4 and MMP13), and anabolic markers (Aggrecan) were detected by Western blotting and cell immunofluorescence. Figure 38-41As shown, in the nucleus pulposus cells transfected with RELA (P65) SiRNA, after RELA (P65) was knocked out, the inhibitory effect of sansin on nucleus pulposus cell inflammation was no longer significant.

[0103] These findings collectively indicate that RELA (P65) is essential for the regulation of sansin-mediated inflammation in nucleus pulposus cells.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of sansin in the preparation of drugs for the treatment of intervertebral disc degeneration.

2. The use according to claim 1, It is characterized in that Sancin inhibits TNF-α-mediated nucleus pulposus inflammatory response and reduces the production of inflammatory factors induced by TNF-α, including IL-1β, IL-6 or COX-2.

3. The use according to claim 1, It is characterized in that Sansin reduces the production of matrix-degrading enzymes, including MMP-13 or ADAMTs4.

4. The use according to claim 1, It is characterized in that Sansin reverses TNF-α-mediated repression of anabolic genes including one or both of ACAN or Col-2.

5. Application of sansin in the preparation of drugs for preventing intervertebral disc degeneration.

6. A drug for preventing and / or treating intervertebral disc degeneration, It is characterized in that The medicine contains sanshinol.

7. The drug according to claim 6, It is characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The drug according to claim 6, It is characterized in that The medicine is an injection.

9. The drug according to claim 8, It is characterized in that The injection includes injection additives in addition to the diluent.

10. The drug according to claim 8, It is characterized in that The injection is administered via intraperitoneal injection.

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