Application of pulsatilla saponin B4 in preparation of medicine for treating intervertebral disc degeneration and intervertebral disc-derived pain

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and discloses application of pulsatilla saponin B4 in preparation of a medicine for treating intervertebral disc degeneration and discogenic pain. The pulsatilla saponin B4 is used for preparing the medicine for treating the intervertebral disc degeneration and the discogenic pain, the intervertebral disc degeneration can be treated, the discogenic pain can be relieved, and the effect is good. The pulsatilla saponin B4 disclosed by the invention is used for preparing the medicine for treating intervertebral disc degeneration and intervertebral disc-derived pain, and the pulsatilla saponin B4 has small influence on cell activity, has no cytotoxicity and is good in medicine safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and is specifically used as a kind of saponin B4 in the preparation of drugs for treating intervertebral disc degeneration and disc-derived pain. Background Art

[0002] Low back pain is a common disease in spinal surgery. The average prevalence of low back pain is high. And the number of people disabled by leg pain is also increasing. For a long time, traditional Chinese medicine has been a major folk remedy for low back pain. However, to date, Chinese medicine has the disadvantages of unclear active substances and unclear mechanisms of action in the context of modern medicine, and lacks objective and scientific evaluation methods and standard specifications for the effectiveness and safety of clinical applications. Therefore, improving the scientific research and innovation capabilities of the Chinese medicine industry and shaping a complete new scientific research system are important goals and strategic directions for achieving the modernization of Chinese medicine.

[0003] Intervertebral disc degeneration is an important cause of low back pain. Studies have shown that disc-related low back pain accounts for 40% of the total number of patients with low back pain. Drug therapy is the most common medical treatment for intervertebral disc degeneration (IDD). IDD and IDH are controlled through various painkillers, nerve nutrition, cartilage nutrition, etc. In clinical practice, sodium aescinate, diuretics and osmotic drugs can also be used to reduce nerve root edema and promote cartilage tissue growth. Various blood-activating, blood-stasis-removing, muscle-relaxing and collateral-activating drugs and plasters have a certain effect on relieving low back muscle pain. In clinical practice, steroid drugs can also be injected directly near the injured nerve root to quickly reduce swelling and relieve pain.

[0004] However, various painkillers, nerve nutrition, and cartilage nutrition can only be used to control the condition of IDD and prevent it from getting worse. They also have significant side effects. For example, the painkiller meloxicam often causes gastrointestinal irritation such as nausea, vomiting, anorexia and other adverse reactions. Long-term use of the muscle relaxant drug eperisone hydrochloride tablets will cause side effects such as limb weakness and drowsiness, while the nerve nutrition drug methylcobalamin tablets may cause allergic reactions such as rashes and other adverse reactions. Although other drugs can reduce nerve root edema and promote the growth of cartilage tissue, they need to be taken for a long time to be effective. Hormone injections only have a short-term effect on nerves and their surrounding tissues, and cannot fundamentally repair the intervertebral disc. At the same time, long-term injections will also bring more serious side effects, so there is a need for better alternative treatment drugs. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide the application of saponin B4 in the preparation of drugs for treating intervertebral disc degeneration and disc-derived pain.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] Application of saponin B4 in the preparation of drugs for the treatment of intervertebral disc degeneration and disc-derived pain.

[0008] The molecular formula of the pulsatilla saponin B4 is C 59 H 96 O 26 , molecular weight is 1221.38, structure is

[0009]

[0010] The medicine contains the ingredient pulsatilla saponin B4.

[0011] The dosage of saponin B4 of the present invention is 1-55umol / L in cells and 100-1000umol / L in rat intervertebral discs.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] 1) The present invention uses pulsatilla saponin B4 for preparing a drug for treating intervertebral disc degeneration and disc-induced pain, which can treat intervertebral disc degeneration and relieve disc-induced pain (such as low back pain and neck pain caused by intervertebral disc degeneration).

[0014] 2) The saponin B4 of the present invention is used to prepare drugs for treating intervertebral disc degeneration and disc-derived pain. The saponin B4 has little effect on cell activity, is non-cytotoxic, and has good drug safety.

[0015] The saponin B4 of the present invention has a significant therapeutic effect in treating disc degeneration and relieving disc-derived pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effects of AB4 on cell activity; A: The effects of AB4 concentration changes on the activity of normal nucleus pulposus cells; B: The effects of AB4 concentration changes on the activity of nucleus pulposus cells induced by IL-1β inflammation;

[0017] Figure 2 Pulsatilla saponin B4 inhibits the expression of cartilage markers in nucleus pulposus cells in degenerative intervertebral discs; A: mRNA level of SOX-9 gene in nucleus pulposus cells; B: mRNA level of Collagen-II gene in nucleus pulposus cells; C: mRNA level of ACAN gene in nucleus pulposus cells, p<0.05;

[0018] Figure 3 Comparison of histopathological changes in rat intervertebral disc histopathological changes (HE staining, MASSON staining, crocodile solid green staining, × 400);

[0019] Figure 4These are the experimental results of scutellaria saponin B4 in relieving discogenic pain; AC: The stimulation pressure at which positive reactions were observed in the Von Frey test on the third, seventh and fourteenth days after administration in the control group and the experimental group, p<0.05; DF: The stimulation time at which positive reactions were observed in the Hargreaves test on the third, seventh and fourteenth days after administration in the control group and the experimental group, p<0.05. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific examples, but the embodiments of the present invention are not limited thereto. The human nucleus pulposus cells (HNPC) used in the examples were extracted, isolated and cultured from intraoperative specimens of the Department of Spine Surgery of Guangdong Provincial People's Hospital.

[0021] Example 1

[0022] A control group was established to measure the effect of AB4 (Pulsatilla saponin B4) administration concentration changes on the cell activity of normal cells, and an experimental group was established to measure the effect of AB4 administration concentration changes on the cell activity of the IL-β-induced intervertebral disc nucleus pulposus cell degeneration model. Multiple AB4 concentration gradients were set, and through CCK-8 experiments, in the presence of an electron coupling reagent, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt) reacted with dehydrogenase in the mitochondria of cells to generate an orange-yellow formazan product (formazan), the color depth of which is proportional to the cell activity, and the OD value was measured at a wavelength of 450nm using an enzyme marker to reflect the cell activity. The experimental results showed that the concentration change of AB4 had little effect on the cell activity of the control group, and there was basically no cytotoxicity or other adverse effects. After AB4 was added to the IL-β-induced degenerative cell model in the experimental group, the cell activity increased compared with the inflammatory cell model without AB4, indicating that the established AB4 dosage concentration can increase cell activity within the appropriate concentration range, and it was found that the cell activity when the AB4 dosage concentration was 50 μM was higher than that when the dosage concentration was 0.5 μM, 1 μM and 10 μM, indicating that the change in AB4 dosage concentration has a concentration effect on the cell activity of the above-established inflammatory cell model, and therefore the appropriate dosage concentration range was determined.

[0023] IL-1β induction is a method to induce disc degeneration and pain models.

[0024] Test results such as Figure 1 shown.

[0025] Example 2

[0026] Well-grown human nucleus pulposus cells were randomly divided into a blank control group, an AB4 group, an IL-1β group, and an IL-1β+AB4 group. The blank control group was not treated with any treatment, the AB4 group was treated with 10μΜ AB4, the IL-1β group was treated with 10ng / ml IL-1β, and the IL-1β+AB4 group was treated with 10ng / ml IL-1β and AB4 at concentrations of 50μΜ, 10μΜ, and 1μΜ, respectively. Three days after administration, total RNA was extracted from the treated cells, and the RNA concentration and purity were detected by Nano-drop; reverse transcription was performed into cDNA, and the mRNA levels of SOX-9, Collagen-II, and ACAN genes, markers of intervertebral disc degeneration, were detected by qRT-PCR technology. Take a 0.2mL PCR tube, prepare the reaction system, prepare 3 tubes for each reverse transcription product, pre-denature at 95℃ for 5min, denature at 95℃ for 10min, anneal at 60℃ for 30s, extend at 72℃ for 30s, and cycle for 40 times. The data were processed by the ΔΔCt method, and 2-ΔΔCt represents the relative expression amount of mRNA of the target gene.

[0027] The results of qPCR showed that IL-1β downregulated the expression of ACAN and SOX-9 at the mRNA level. The above-mentioned SOX-9, Collagen-II, and ACAN are commonly used marker genes for degeneration of the nucleus pulposus tissue of the intervertebral disc. It is generally believed that the expression is negatively correlated with the degree of intervertebral disc degeneration. The higher the degree of degeneration, the lower the transcription level of the three genes. The results of qPCR showed that the mRNA levels of the three genes in the AB4 group were increased compared with the blank control group, the mRNA levels of the three genes in the IL-1β group were greatly reduced compared with the blank control group, and the mRNA levels of the three genes in all IL-1β+AB4 groups were increased compared with the IL-1β group. At the same time, it was found that the mRNA levels of the three genes in the IL-1β+AB4 group were not much different when the AB4 administration concentration was 1μΜ and 10μΜ, and the mRNA levels of the three genes were significantly increased when the AB4 administration concentration was 50μΜ, indicating that AB4 has a certain concentration effect on the mRNA levels of the above three genes. The above results show that AB4 can inhibit mRNA expression of the degeneration marker gene of intervertebral disc nucleus pulposterior cells.

[0028] Test results such as Figure 2 shown.

[0029] Example 3

[0030] The experimental rats were anesthetized by injection, and a 5μL microsyringe with a reserved length was used to pierce the center of the intervertebral disc, about half the height of the intervertebral disc, and slowly rotated for one circle, and 2μL of 0.05mg / mL IL-1β was injected, and the liquid was kept for 30 seconds until it was fully absorbed and then slowly pulled out to establish an animal inflammation model. A blank control group, a PBS group, an AB4 group, an IL-1β group, and an IL-1β+AB4 group were set up. The blank control group did not receive any treatment, the PBS group only received acupuncture injection of PBS, the AB4 group received AB4 treatment at a concentration of 1000μmol / L, the IL-1β group received IL-1β treatment at 10ng / mL, and the IL-1β+AB4 group received IL-1β treatment at 10ng / mL and AB4 treatment at a concentration of 1000μmol / L. After 14 days of administration, the intervertebral discs of the rats were taken for pathological sections and HE staining, safranin fast green staining, and MASSON staining were performed to observe the morphology of the intervertebral discs of the rats. The results of three sections staining showed that the boundary between the nucleus pulposus and the annulus fibrosus of the blank control group rats was clear, the annulus fibrosus structure was intact, and the nucleus pulposus cells were neatly and evenly arranged; the boundary between the nucleus pulposus and the annulus fibrosus of the PBS group rats was blurred, the annulus fibrosus was disordered and there were gaps, the nucleus pulposus cells were aggregated and shrunken, and the matrix was reduced; the annulus fibrosus structure and nucleus pulposus morphology and arrangement of the AB4 group rats were similar to those of the PBS group rats, indicating that AB4 had no damaging effect on the cells; compared with the PBS group, the annulus fibrosus of the IL-1β group rats had serious cracks, the number of nucleus pulposus cells was significantly reduced, and the degree of degeneration was serious; the intervertebral disc lesions of the IL-1β+AB4 group rats were less than those of the IL-1β group, and the annulus fibrosus structure and nucleus pulposus morphology and arrangement tended to be normal. The above results show that AB4 has an anti-disc degeneration effect.

[0031] Test results such as Figure 3 shown.

[0032] Example 4

[0033] The experimental rats were anesthetized by injection, and a 5μl microsyringe with a reserved length was used to pierce the center of the intervertebral disc, about half the height of the intervertebral disc, and then slowly rotated for one circle. 2μl of 0.05mg / ml IL-1β was injected, and the syringe was left for 30 seconds until the liquid was fully absorbed and then slowly withdrawn to establish an animal inflammation model. A blank control group, a normal rat intervertebral disc model control group injected with PBS, a control group injected with 1000 μmol / L AB4, and a control group of the rat intervertebral disc inflammation model established above were set up. Experimental groups of the rat intervertebral disc inflammation model established above with AB4 administration concentrations of 1000 μmol / L were set up. On the third, seventh, and fourteenth days after administration, Von Frey experiments were performed: using a fiber with a determined stimulation pressure in grams to stimulate the rat tail and record the stimulation pressure corresponding to the positive reaction; Hargreaves experiments were performed: using a heat source 10 cm away from the rat tail to stimulate the rat tail, and recording the time when the rat licked the tail, shook the tail, and other positive reactions occurred. If no positive reaction occurred after 40 seconds of stimulation, 40 seconds was recorded as the upper limit to prevent damage. The experiment found that compared with the normal rat intervertebral disc model, the stimulation pressure and stimulation time of the rat intervertebral disc inflammation model were reduced, showing an increased sensitivity to pain. Compared with the model group, the stimulation pressure and stimulation time of the positive reaction in the experimental group increased, preliminarily proving that AB4 can relieve discogenic pain.

[0034] Test results such as Figure 4 shown.

Claims

1. Application of pulsatilla saponin B4 in the preparation of drugs for the treatment of intervertebral disc degeneration and discogenic pain.

2. The use according to claim 1, characterized in that: The molecular formula of the pulsatilla saponin B4 is C 59 H 96 O 26 , molecular weight is 1221.38, structure is 3. A drug for treating intervertebral disc degeneration and intervertebral disc-related pain, characterized in that: The medicine contains the ingredient pulsatilla saponin B4.

4. The drug for treating intervertebral disc degeneration and intervertebral disc-related pain according to claim 3, characterized in that: The drug is a drug for inhibiting the mRNA expression of intervertebral disc nucleus pulposus cell degeneration marker genes and relieving intervertebral disc-derived pain.

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