CN110123868B - Application of scorpion-containing traditional Chinese medicine composition in preparation of medicine for treating rheumatoid arthritis bone erosion - Google Patents

Abstract

The invention discloses an application of a scorpion-containing traditional Chinese medicine composition in preparing a medicine for treating rheumatoid arthritis bone erosion. The applicant researches and discovers that the Chinese medicinal composition for dispelling wind and removing stasis can inhibit the expression of IL-6 and RANKL in various cells at the joint part of a CIA mouse, increase the expression of osteoprotegerin OPG, reduce the number of osteoclasts, increase bone density, reduce bone erosion of the CIA mouse, effectively relieve swelling of the joint of the CIA mouse and reduce damage of the synovial membrane of the joint, and has a pharmaceutical application prospect for related diseases.

Description

Application of scorpion-containing traditional Chinese medicine composition in preparation of medicine for treating rheumatoid arthritis bone erosion

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of a scorpion-containing traditional Chinese medicine composition in preparation of a medicine for treating rheumatoid arthritis bone erosion (hereinafter referred to as wind-dispelling and stasis-removing traditional Chinese medicine composition and pharmaceutical application thereof).

Background

Rheumatoid Arthritis (RA) is called immortal cancer (Roaming et al, the category of cancer belonging to 'phlegm of Bi' in traditional Chinese medicine. the journal of basic Chinese medicine 2015,21(01):14-15), is a systemic autoimmune disease mainly manifested by erosive polyarthritis, and is a common disease and a difficult and complicated disease with high disability rate and great harm, wherein RA is manifested by symmetrical arthritis at the early stage and is stiff and malformed at the late stage. Worldwide, there is 0.5-1% of adult diseases, with female more than male (Chung IM, et al, Rheumatoid Arthritis: The strand from Research to Clinical practice. International journal of molecular sciences, 2016; 17). The prevalence rate of RA in China is about 0.3% -0.42%, more than 500 million people suffer from RA symptoms for a long time, and the prevalence of RA is more serious especially in underdeveloped areas. Patients often lose their ability to work due to illness resulting in increased economic difficulties in the family and social burden. Therapeutic agents for RA are mainly disease-modifying anti-rheumatic drugs (DMARDs) for alleviating disease conditions (Ichikawa N, et al, disease-modifying anti-rheumatic drugs, Clin Calcium,2012,22(2):215-221)), and are classified into two classes, i.e., non-biological agents (tdrdms) and biological agents (bdrdms). tDMARDs are characterized by slow onset of action, low cost, bone destruction still proceeding after RA inflammation control, and difficulty in inhibiting the progress of bone destruction. The bDMARDs have quick response, but very high cost (about 10 thousands of medical cost per year), and are effective at a single target point, so that the bDMARDs cannot be suitable for complicated conditions of all patients, and the curative effect of a part of patients is not ideal; and are unacceptable to most patients because of their potential infection-inducing side effects (Dimitroulas T, et al. biologic therapeutics and system bone loss in rheumoid arthritis. autoimmun Rev,2013,12(10): 958-966). At present, DMARDs and methotrexate are mostly used for clinically treating RA (Watsenberg S, et al, very low-dose-therapy in early rheumatoid arthritis patients radiographical progress over two years: a multicenter, double-blind, plasma-controlled trial, arthritis and rheummation 2005; 52:3371-3380), RA is treated only from the symptom relief angle, diseases cannot be completely cured, patients need to take medicines for a long time, and RA medical cost becomes a heavy burden of the national health care. Therefore, the enhancement of the research on RA drugs becomes one of the problems to be solved urgently in the prevention and treatment system of serious diseases in China.

Bone erosion is a major feature of RA. Modern studies have found that RA bone erosion is associated with the entire course of RA, and that bone loss begins early in the course of RA, often in the first 2 years of the course, with the disease in active phase, and occurs most rapidly. And once the bone is destroyed, the pathological change is meant to enter an irreversible stage, and the local bone erosion of the joint of the RA patient can cause the joint deformity and the function loss with the lapse of time, and the disease causes disability for 5 to 10 years, so that the labor capacity loss ratio of the patient reaches 33 to 39 percent. At present, the clinical treatment standard of RA is only to relieve symptoms, and no good radical treatment method exists. The over-differentiation of osteoclast is an important reason for RA bone erosion, RANKL is a factor for directly inducing osteoclast generation, NF-kappa B is a key transcription factor for RANKL to induce osteoclast differentiation and maturation, after RANK on the surface of osteoclast precursor cell is combined with RANKL, IKK is activated, so that phosphorylation of Ikappa B alpha is caused, further ubiquitin-linked ubiquitin is performed, and degradation is performed by protease to form free NF-kappa B (P65), transcription of osteoclast-related genes (cathepsin K, tartrate-resistant acid phosphatase and the like) is started after nuclear entry (Lorenzo J.the many of osteoenclast activity.journal of clinical information 2017; 127: 2530-. Osteoprotegerin (OPG), a key Role in the regulation of bone metabolism, can highly bind to RANKL to block the binding of RANK to RANKL, and has the main functions of inhibiting the differentiation and activation of osteoclasts, promoting osteoclast apoptosis, and inhibiting bone resorption (Corrado A, et al. Osteoblast roll in Rheumatic diseases. int J Mol Sci.2017Jun 15; 18 (6)). In the inflammatory state of RA, the release of various inflammatory factors such as TNF-a, IL-6, IL-17, etc. is increased, which promotes the differentiation of osteoclasts, increases osteoclast activity, limits osteoblast activity, causes disturbance of bone metabolism, and leads to bone erosion (1.Abdel Meguid MH, et al.relationship of interleukin-6 in rhematoid arthritis bone loss and structural bone mass index. Rheumatoid protein. 2013 Mar; 33(3) KL 697-703.2.Luo G, et al.TNF-a and RANmo bone morphogenesis by regulating RANK video of NF kappa B tissue. molecular ligand proteins 2018; 17: 05-6611.3. Gamma R, gamma. CSF-2. interleukin 20117, Mi III-23, gamma-23. gamma. 12. gamma. 23. gamma. 12. gamma. 12. gamma. 12. gamma. medium proteins, 23. gamma. 12. gamma. 12. gamma. 12. gamma. 12. gamma. proteins, gamma. 12. gamma. 12. proteins, 23. gamma. 12. gamma. proteins, gamma. 12. gamma. proteins, gamma. 12. gamma. 12. gamma. 12. gamma. 12. gamma. 12. gamma. proteins, gamma. 12. gamma. proteins, gamma. 12. gamma. proteins, gamma. 12. proteins, gamma. proteins, gamma. 12. gamma. proteins, gamma. 12. proteins, gamma. gamma, bone erosion is the bottleneck of the current RA treatment, osteoclast over differentiation is an important reason for causing RA bone erosion, RANKL/NF-kB/NFATc 1 is a key signal path for mediating osteoclast differentiation, and multiple proinflammatory cell factors promote osteoclast differentiation, so that RA bone erosion is caused by multiple ways and mechanisms and is difficult to achieve a better effect by the treatment of a single-target-point medicament.

Currently, anti-rheumatoid drugs (DMARDs) are clinically used for mainly relieving joint swelling and inflammation of patients, but the effect of improving bone erosion is not obvious. Antibody drugs that specifically target RANKL, while able to slow disease progression, have no established efficacy for bone erosion.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the prior art, the invention provides a traditional Chinese medicine composition for dispelling wind and resolving masses, application thereof in preparing medicines for treating bone erosion related diseases, and application thereof in preparing medicines for treating bone erosion of rheumatoid arthritis and rheumatoid arthritis.

The technical scheme is as follows: the invention discloses a traditional Chinese medicine composition for dispelling wind and removing stasis, which comprises the following components: scorpio, Scolopendra, radix astragali, and semen Sojae Atricolor coat.

Preferably, the wind-dispelling and stasis-removing traditional Chinese medicine composition (hereinafter referred to as the wind-dispelling and stasis-removing formula) is prepared from 1.5g of scorpion, 1.5g of centipede, 20g of astragalus membranaceus and 30g of black bean coat in a compatibility manner.

The invention also discloses application of the wind-dispelling and stasis-removing traditional Chinese medicine composition in preparation of a medicine for treating bone erosion.

The invention also discloses application of the wind-dispelling and stasis-removing traditional Chinese medicine composition in preparation of a medicine for treating rheumatoid arthritis bone erosion.

The invention also discloses application of the wind-dispelling and stasis-removing traditional Chinese medicine composition in preparation of a medicine for treating rheumatoid arthritis.

The research shows that the Chinese medicinal composition for dispelling wind and removing stagnation can inhibit the expression of IL-6 and RANKL in various inflammatory cells at joint parts, increase the expression of Osteoprotegerin (OPG), increase bone density and reduce the number of osteoclasts, effectively relieve the swelling of joints of a mouse CIA, reduce the damage of joint synovium and reduce the bone erosion of the mouse CIA.

The compatibility formula: the wind-dispelling and stasis-dispersing formula consists of four medicines of scorpion, centipede, astragalus and black bean coat. The scorpion has pungent taste and mild property, enters liver meridian, has the functions of calming wind, relieving spasm, counteracting toxic pathogen, dissipating stagnation, dredging collaterals and relieving pain, is good at moving and penetrating tendons and bones, and is an essential medicine for treating chronic arthralgia and obstinate arthralgia. Wu Gong is pungent in flavor and slightly warm in nature, enters liver and heart meridians, has special actions of relieving spasm and removing toxicity, and can unblock meridians to extinguish liver wind. The two herbs are mutually reinforced and complement each other, and they are the monarch drugs, and they can search wind and flee to the zang-fu organs and the meridians and collaterals, all of which can be developed for the accumulation of qi and blood. The astragalus root is the minister, the taste is sweet and warm, the viviparous is cool, and the book of materia medica works, the cloud: astragalus root, radix astragali, also has the essential herbs of tonifying lung and spleen, checking sweating, dispelling wind and removing toxicity. The specific functions of Huang Qi in this book are to strengthen the body and defend the body, astringe the sweat, promote the secretion of saliva or body fluid, nourish the muscles and bones, and promote the growth of tissue. For patients with arthralgia, if the disease is lingering, the qi will be consumed and yin will be damaged, so that on the one hand, astragalus root can tonify middle-jiao to strengthen body resistance, nourish muscles and bones, promote tissue regeneration and strengthen the effect of dredging collaterals; on the other hand, the medicine can restrict the insect drugs from being too pungent, dispersing and warming to dredge, and alleviate the defects of invasion and qi consumption of the insect drugs. Black bean coat, sweet in taste and neutral in nature, enters blood system and enters kidney meridian. In Ben Cao Quzhen, it is said that "it can cure water and relieve distension, descend qi to control wind-heat, activate blood and remove toxicity". Therefore, the traditional Chinese medical doctor Zhongyanqiqifrequently uses to detoxify, dispel wind, nourish blood and induce diuresis, and the medicine can not only help the astragalus root to strengthen and transport middle-jiao spleen and stomach, make body fluid and blood active and make evil remove but not damage body resistance; can also be used in combination with radix astragali to assist in the preparation of scorpion and centipede for preventing yin and blood damage. The whole formula has the functions of tonification and purgation, and the compatibility is proper.

According to the clinical manifestations of RA, TCM classifies RA into the category of arthralgia syndrome. Therefore, the use of the Chinese medicinal composition for treating RA accords with the theoretical thought of treatment based on syndrome differentiation of traditional Chinese medicine, and is inherited and developed again for the traditional Chinese medicines.

Has the advantages that: compared with the existing clinical use of anti-rheumatoid drugs (DMARDs), the medicine mainly relieves the joint swelling and internal inflammation of patients, but has an insignificant improvement effect on bone erosion; antibody drugs that specifically target RANKL, while able to slow bone erosion, have no significant effect on inflammation and joint swelling (Kamijo S, et al. Electron analysis of bone loss in collagen-induced arthritis by neutral anti-RANKL monoclonal antibody. Biochemical and biological research communications.2006; 347: 124: 132). The wind-dispelling and stasis-removing traditional Chinese medicine composition can relieve joint swelling and inflammation of CIA mice, can obviously improve bone erosion, has double regulation effects, and is expected to be developed into new drugs for related diseases.

Drawings

FIG. 1 is a wind-dispelling and nodulation-dissipating prescription for relieving swelling of joints of CIA mice;

FIG. 2 shows that the wind-dispelling and stasis-removing formula improves the joint destruction of CIA mice;

FIG. 3 shows that the wind-dispelling and stasis-removing formula inhibits the decrease of bone density of ankle joints of CIA mice;

FIG. 4 shows that the wind-dispelling and stasis-removing formula inhibits inflammatory cell infiltration of joints, cartilage and synovial membrane damage of CIA mice;

FIG. 5 shows that the wind-dispelling and stasis-removing formula reduces the number of osteoclasts in the joints of a CIA mouse;

FIG. 6 shows that the wind-dispelling and stasis-removing prescription inhibits the expression of RANKL in joints of a CIA mouse;

FIG. 7 shows that the wind-dispelling and stasis-removing formula promotes the expression of OPG (osteoprotegerin) in joint bone of a CIA mouse;

FIG. 8 shows that the wind-dispelling and stasis-removing formula inhibits the expression of IL-6 at the joint part of a CIA mouse;

in which the arrows in figures 5-8 indicate positively stained cells.

Detailed Description

The present application will be described in detail with reference to specific examples.

The source of the drug is as follows: methotrexate, scorpion and centipede decoction pieces are all sourced from pharmacies of Chinese and western medicine combined hospitals in Jiangsu province.

Preparing a wind-dispelling and stasis-removing formula: taking scorpion decoction pieces, crushing into coarse powder, drying at 50-60 ℃, crushing again, sieving by a 80-mesh sieve, and taking 1.5g for later use; taking centipede decoction pieces, removing heads and feet, crushing, sieving with a 80-mesh sieve, and taking 1.5g for later use; pulverizing Scorpio and Scolopendra at a weight ratio of 1:1 with 80 mesh sieve, and mixing. 20g of astragalus and 30g of black bean coat are decocted, and then the scorpion centipede powder is added to be evenly mixed to prepare the medicine with the corresponding concentration for animal experiments. Mouse RA model modeling

60 healthy male DBA/1J mice (purchased from the institute of Biotechnology, university of Nanjing) at 6-7 weeks were divided into 10 normal groups and 50 modeling groups by a complete randomized cohort method. The model group mice are all modeled by a method of twice immunization of bovine type II collagen. For the first immunization, complete Freund's adjuvant (purchased from Chondrex) and bovine type II collagen (purchased from Chondrex) were completely emulsified in a sterile ice-bath tube using a phacoemulsification machine, and the emulsion was aspirated using a 1mL syringe, and a needle tip was subcutaneously inserted into each mouse at a distance of 2cm from the caudal root while avoiding the caudal vein, and 100uL was injected into the caudal root at a distance of 0.5cm from the caudal root by inserting 1.5cm in parallel into the caudal root. On day 21, a second booster immunization was performed to emulsify collagen and Freund's incomplete adjuvant, and 100uL of emulsion was injected at a distance of 3cm from the caudal root, 1.5cm from the subcutaneous tissue, and 1.5cm from the caudal root.

Grouping and administration of RA animals

After secondary immunization, when the incidence rate of the molded mice reaches nearly 80%, the CIA mice which are successfully molded are divided into three groups: a model control group, a wind-dispelling and stasis-removing prescription group and a methotrexate positive medicine group, and a normal control group is set. The administration is started the next day, the group of medicine for searching wind and resolving masses is subjected to intragastric administration (containing 9.72g/Kg of crude drug), the group of methotrexate tablets (0.9g/Kg, 2 times per week) is subjected to intragastric administration, the model control group and the normal control group are subjected to distilled water with the same volume, and the samples are killed and collected after 28 days of continuous administration.

(1) The general conditions of DBA/1J mice, including hair, mental state, activity, muscle, and joint, were observed several days prior to the experiment, and mice in good condition were selected for modeling and dosing. Mice body weight, joint changes, and death were recorded at the beginning of the first immunization and weekly. Scoring and recording the disease degree of the limbs of the arthritic mice: from the onset of the disease of the mice after the second immunization, the disease condition of the mice was recorded by four-limb scoring every three days. Four limb arthritis scoring criteria (4 points system): 0 minute: no evidence of redness and swelling; 1 minute: erythema and mild swelling localized to the midfoot (tarsal bones) or ankle joint (a slight red swelling of one toe); and 2, dividing: erythema and mild swelling spread from the ankle to the midfoot (with 2 or more toes red); and 3, dividing: erythema and moderate swelling spread from the ankle to the metatarsal joints (redness of the ankle or hip); and 4, dividing: erythema and severe swelling included ankles, feet and toes (all joints and toes are red and swollen and unable to bend). All limbs were scored individually, so the highest score per mouse was 16.

(2) Scanning mouse feet and joint structures of different groups by a Micro-CT instrument, carrying out toe imaging analysis on live mice under the Micro-CT instrument after anesthetizing the live mice to observe the joint structures of the mouse feet and the like, comparing the deformation and damage degrees of joints of the mice of each group, and comparing the bone density of the joints of the mice of each group by bone density analysis software.

(3) Taking ankle joints and synovial tissues of each group of mice, H & E staining, and observing pathological changes such as joint synovial lining cell proliferation, lining underlayer inflammatory cell infiltration, cartilage destruction degree and pannus formation under a light microscope; (scores were scored according to degree: 0: normal; 1: mild focal infiltration, minimal inflammatory cell infiltration or mild edema; 2: moderate infiltration; 3: severe inflammatory infiltration but no vascularization and cartilage destruction; 4: very severe infiltration forming vascularization andor cartilage, bone erosion).

(4) The ankle joints of mice in each group are taken, fixed and decalcified, embedded in paraffin, sliced, and subjected to TRAP staining to observe the change of the number of the osteoclasts in the joints in each group.

(5) Taking paraffin sections of ankle joints of mice of each group, and detecting the expression conditions of IL-6, RANKL and OPG of joints of each group through immunohistochemistry.

Secondly, the results

1. The wind-dispelling and node-resolving formula is used for relieving the swelling of the joints of the CIA mice: as shown in figure 1, compared with red swelling of toes of the mice in the CIA control group, swelling of feet gradually spreads to ankle joints, and the red swelling of the toes and the ankle joints of the CIA mice is obviously reduced after the wind-dispelling and stasis-removing treatment.

2. The wind-dispelling and stasis-removing formula improves the joint destruction degree of the CIA mice: and scanning the structures of the paw and each joint of the mice of different groups by using a Micro-CT instrument. As shown in FIG. 2, the bone surface of the normal control group mice was smooth and the structure was intact; and the bone erosion of the mice in the model control group is serious, and some mice even form cavities. After the treatment of the wind-searching and stagnation-eliminating formula, the bone surface erosion degree of the CIA mice is obviously improved, which shows that the wind-searching and stagnation-eliminating formula can effectively delay the bone erosion of arthritis.

3. The wind-dispelling and stasis-resolving formula is used for increasing the bone density of the ankle joint of the CIA mouse: and (3) taking the ankle joints of each group of mice, and analyzing and determining the bone density of the ankle joints of each group of CIA mice after micro-CT scanning. As can be seen from fig. 3, bone density values of the CIA control mice were significantly reduced compared to the normal mice. The bone density value of the mice in the wind-dispelling and stasis-removing formula treatment group is obviously increased compared with that of the mice in the model group. The wind-dispelling and stasis-resolving formula can increase the bone density of the CIA mice and improve bone erosion.

4. The wind-dispelling and stasis-removing formula is used for inhibiting osteoarthritis cell infiltration and cartilage and synovial membrane damage of a CIA mouse: and (3) taking the ankle joints of each group of mice, fixing and decalcifying the ankle joints, embedding paraffin, slicing, and carrying out H & E staining to observe the change of the ankle joints and synovium of each group. As shown in figure 4, compared with the normal mouse, the ankle joint structure is complete, the synovium is smooth, no inflammatory cell infiltrates, the synovium is thickened, the cartilage is damaged, the bone is eroded, the joint structure is seriously damaged and deformed due to the infiltration of a large number of inflammatory cells in the joint cartilage and the synovium of the CIA control mouse, and the membrane proliferation and inflammatory cell infiltration, cartilage destruction and bone erosion degree of the joint of the CIA mouse are obviously reduced by the wind-searching and nodule-removing formula.

5. The wind-dispelling and stasis-resolving formula reduces the number of osteoclasts in joint tissues of the CIA mice: the ankle joints of mice in each group are taken, fixed and decalcified, embedded in paraffin, sliced, and subjected to TRAP staining to observe the change of the number of the osteoclasts in the joints of the mice in each group. As shown in FIG. 5, compared with the normal control mouse, the ankle joint of the mouse has almost no visible wine red colored osteoclast, a large amount of osteoclast is found at the ankle joint of the CIA mouse, the joint structure is seriously damaged and deformed, and the osteoclast number of the wind-searching and stasis-removing formula treatment group is obviously reduced compared with that of the CIA control group.

6. The wind-searching and stagnation-removing prescription is used for inhibiting the expression of RANKL in joint cells of a CIA mouse, and the RANKL expression in the joint cells of the mouse is detected by taking paraffin sections of ankle joints of various groups of mice and adopting an immunohistochemical technology. As shown in FIG. 6, compared with low-expression RANKL in ankle joints of normal mice, the mice in the CIA control group highly express RANKL, and the expression of RANKL in joint parts is obviously reduced by the wind-searching and node-dissipating prescription.

7. The wind-dispelling and stagnation-removing formula is used for increasing the expression of Osteoprotegerin (OPG) at joint parts of a CIA mouse, and the OPG expression of joints of each group is detected by taking paraffin sections of ankle joints of each group of mice and carrying out immunohistochemistry. The results are shown in fig. 7, and compared with the expression of OPG of the mouse in the CIA control group, the expression of OPG of the mouse in the treatment group of the wind-searching and stasis-resolving formula is increased, which indicates that the expression of OPG of the joint of the CIA mouse can be increased by the wind-searching and stasis-resolving formula.

8. The effect of the wind-searching and stagnation-eliminating formula on the expression of IL-6 in the joint cells of the CIA mice is that the ankle joint paraffin sections of all groups of mice are taken, and the expression of IL-6 is detected by immunohistochemistry, and the result is shown in figure 8.

Claims (2)

1. The application of a scorpion-containing traditional Chinese medicine composition in preparing a medicine for treating rheumatoid arthritis bone erosion is characterized by being prepared from the following components: 0.5-2g of scorpion, 0.5-2g of centipede, 15-25g of astragalus root and 25-35g of black bean coat, and the specific preparation method comprises the following steps: pulverizing Scorpio decoction pieces into coarse powder, oven drying at 50-60 deg.C, pulverizing, and sieving with 80 mesh sieve; taking centipede decoction pieces, removing heads and feet, crushing, and sieving with a 80-mesh sieve for later use; taking the scorpio and the centipede which are crushed by passing through an 80-mesh sieve, mixing uniformly according to the weight ratio of 1:1, decocting the astragalus and the black bean coat, adding the scorpio and the centipede powder, mixing uniformly, and preparing the medicine with the corresponding concentration.

2. The use of the Chinese medicinal composition containing scorpion as claimed in claim 1 for preparing a medicament for treating rheumatoid arthritis bone erosion, wherein the medicament comprises scorpion 1.5g, centipede 1.5g, astragalus root 20g and black soya bean 30 g.

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