New application of wind-transforming pill and analysis method of wind-transforming pill

Abstract

The invention discloses a new application and an analysis method of wind-transforming pills. The new application is the application of the wind-transforming pill in preparation of drugs for treating rheumatoid arthritis. According to the analysis method, the action mechanism of the wind-transforming pill for treating rheumatoid arthritis is analyzed by utilizing network pharmacology and a molecular docking technology; comprising the following five steps: screening active ingredients and potential targets, screening diseases and intersection targets, constructing a PPI network and screening core targets, performing GO enrichment analysis and KEGG pathway analysis, and constructing a network of'medicine-active ingredients-diseases-pathway-targets'. The invention develops a new application of the wind-transforming pill in treating rheumatoid arthritis. When the wind-transforming pill is used for treating rheumatoid arthritis, the choices for treating diseases are increased, the living quality of patients is improved, the disease burden of the patients is reduced, and the public health level is improved; and the cost of long-term medical expenses is reduced, the medical care expenditure is controlled, and the efficiency of a health system is improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a new use of Huafengdan and an analysis method thereof. Background Art

[0002] Huafengdan was first created in the 17th century and is one of the classic prescriptions in Guizhou. It is included in the Internal Medicine Volume of the National Compilation of Chinese Patent Medicine Standards. The prescription is composed of more than ten kinds of precious Chinese medicines, including mother medicine, borneol, and silkworm. The mother medicine is fermented from various toxic Chinese medicines such as white aconite, raw pinellia, and raw arisaema. It has the effects of calming wind and stopping spasms, clearing phlegm and opening the orifices. It is used for wind-phlegm obstruction, hemiplegia caused by stroke, epilepsy, facial nerve paralysis, and crooked mouth and eyes.

[0003] At present, the existing functions and indications of Huafengdan do not include the treatment of rheumatoid arthritis. The present invention uses methods such as Elisa to measure serum factors and histopathological analysis to conduct an in vivo pharmacodynamic study on the treatment of rheumatoid arthritis by Huafengdan, and preliminarily proves that Huafengdan has a good therapeutic effect on rheumatoid arthritis, providing a theoretical basis for adding new functions and indications to Huafengdan. At the same time, the application of Huafengdan to treat rheumatoid arthritis increases the choice of treatment, improves the quality of life of patients, reduces the disease burden of patients, and is conducive to improving the level of public health. In addition, the new functions and indications of Huafengdan are conducive to reducing the cost of long-term medical expenses, controlling medical and health care expenditures and improving the efficiency of the health system. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of Huafengdan.

[0005] Another object of the present invention is to provide an analytical method for a new use of Huafeng Dan, and to analyze the mechanism of action of Huafeng Dan in treating rheumatoid arthritis using network pharmacology and molecular docking technology.

[0006] The present invention is achieved by adopting the following technical solutions:

[0007] The new use of Huafengdan described in the present invention is specifically the use of Huafengdan in preparing medicine for treating rheumatoid arthritis.

[0008] The formula of Huafengdan of the present invention is composed of 45.5 parts of mother herb, 40.725 parts of perilla leaves, 18.2 parts of bombyx batryticatus, 9.175 parts of scorpion, 9.175 parts of processed arisaema, 9.175 parts of atractylodes, 6.675 parts of realgar, 18.125 parts of borax, 3.125 parts of croton frost, 0.625 parts of artificial musk, 9.175 parts of borneol, 18.2 parts of gastrodia, 4.575 parts of schizonepeta, 0.825 parts of santalinum, and 6.05 parts of cinnabar.

[0009] The mother medicine of the invention is composed of 126.7 parts of ox bile, 28.2 parts of white aconite root, 28.2 parts of raw pinellia, 28.2 parts of raw arisaema, 28.2 parts of raw chuanwu, 14.1 parts of curcuma and 0.14 parts of Shenqu.

[0010] The analysis method of the new use of Huafeng Dan described in the present invention utilizes network pharmacology and molecular docking technology to analyze the mechanism of action of Huafeng Dan in treating rheumatoid arthritis, including five major steps: screening of active ingredients and potential targets, screening of diseases and intersection targets, construction of PPI network and core target screening, GO enrichment analysis and KEGG pathway analysis, and construction of a "drug-active ingredient-disease-pathway-target" network.

[0011] The screening of active ingredients and potential targets described in the present invention is specifically as follows: the active ingredients of Huafengdan are screened using the PubChem database and the Swiss ADME website, gastrointestinal absorption is high, and at least two of the five drug-like principles (Lipiski, Ghose, Veber, Egan and Muegge) are satisfied if the results are "Yes", and finally the ingredient targets are queried using the SwissTargetPredictio website.

[0012] The screening of disease and intersection targets described in the present invention is specifically as follows: using "rheumatoid arthritis" as a keyword, collecting disease targets in the OMIM, TTD and Gene Cards databases, deleting duplicates to obtain disease targets, and drawing a VENNY diagram to obtain component and disease intersection targets.

[0013] The construction of the PPI network and the screening of core targets described in the present invention are specifically as follows: the intersection targets are imported into the STRING website to construct a PPI network diagram, and the Cytoscape software is used to perform a visualization analysis on it, and then the "CytoNCA" plug-in is used to perform a topological analysis, and the core targets are selected using the degrees of freedom, closeness centrality and betweenness centrality as quantitative indicators.

[0014] The GO enrichment analysis and KEGG pathway analysis described in the present invention are specifically as follows: the intersection targets are imported into the DAVID database, GO functional analysis and KEGG pathway enrichment analysis are performed, and then the top results are selected according to the P value sorting and visualized on the microbial information platform.

[0015] The "drug-active ingredient-disease-pathway-target" network construction described in the present invention is specifically as follows: using Cytoscape software to construct a "drug-active ingredient-disease-pathway-target" network diagram, and selecting components with Degree values ​​≥ 109 as the main therapeutic components.

[0016] The main therapeutic ingredients obtained by selecting the Degree value ≥ 109 in the present invention are new curcuma dione, 9-oxo-11-(3-pentyloxiran-2-YL)undec-10-enoic acid, songguoling, α-linolenic acid, atractylodes lactone III, dihydrosphingosine, Ignavine, coral acanthoate, naked delphinium, and dihydroatisine.

[0017] The present invention has the following advantages:

[0018] The present invention develops a new use of Huafengdan, which is the use of Huafengdan in preparing a drug for treating rheumatoid arthritis. The use of Huafengdan in treating rheumatoid arthritis increases the choice of treatment, improves the quality of life of patients, reduces the disease burden of patients, and is conducive to improving the level of public health; it is conducive to reducing the cost of long-term medical expenses, and is conducive to controlling medical care expenditures and improving the efficiency of the health system. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Component-Disease Venn Diagram

[0020] Figure 2 :PPI network diagram

[0021] Figure 3 :Core target relationship diagram

[0022] Figure 4 :GO function enrichment map

[0023] Figure 5 : KEGG pathway enrichment map

[0024] Figure 6 :Histopathology

[0025] Figure 7 :Micro CT images

[0026] Figure 8 :Expression level of RF in serum

[0027] Fig. 9 : Expression level of BAFF in serum

[0028] Fig.10 :The expression level of TNF-α in serum

[0029] Fig.11 :Expression level of IL-1β in serum DETAILED DESCRIPTION

[0030] The present invention is described in detail below with reference to specific embodiments.

[0031] Example 1 Method for analyzing the mechanism of action of Huafengdan in treating rheumatoid arthritis using network pharmacology and molecular docking technology

[0032] 1 Experimental methods

[0033] 1.1 Data sources are as follows: Table 1:

[0034] Table 1 Database source table

[0035]

[0036] 1.2 Screening of active ingredients and potential targets

[0037] PubChem database and Swiss ADME website were used to screen the active ingredients of Huafengdan. Gastrointestinal absorption was high and at least two of the five drug-like principles (Lipiski, Ghose, Veber, Egan and Muegge) were "Yes". Finally, SwissTargetPredictio website was used to query the ingredient targets.

[0038] 1.3 Screening of diseases and overlapping targets

[0039] Using "rheumatoid arthritis" as the keyword, we collected disease targets from the OMIM, TTD, and Gene Cards databases, deleted duplicates, and obtained disease targets. We drew a VENNY diagram to obtain the intersection targets of components and diseases.

[0040] 1.4 Protein-protein interaction networks (PPI) construction

[0041] The intersection targets were imported into the STRING website to construct a PPI network diagram, and the Cytoscape software was used for visualization analysis. Then, the "CytoNCA" plug-in was used for topological analysis, and the core targets were selected using degrees of freedom (Degree), closeness centrality (Closeness) and betweenness centrality (Betweenness) as quantitative indicators.

[0042] 1.5 Pathway enrichment analysis

[0043] The intersection targets were imported into the DAVID database for Gene ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The top results were selected based on the P-value ranking and visualized on the microbial information platform.

[0044] 1.6 Construction of “drug-ingredient-disease-pathway-target” network diagram

[0045] The Cytoscape software was used to construct a "drug-active ingredient-disease-pathway-target" network diagram. The larger the network node, the stronger the synergistic effect. The ingredients with a degree value ≥ 109 were selected as the main therapeutic ingredients.

[0046] 2 Experimental Results

[0047] 2.1 Components and disease targets

[0048] The active ingredients of Huafengdan were screened through the Swiss ADME website, and 202 therapeutic active ingredients were obtained. 1219 ingredient targets were found from the SwissTargetPredictio website. 7163 disease targets were obtained from the three databases of OMIM, TTD and Gene Cards. A total of 704 ingredient-disease intersection targets were obtained. The results are shown in Figure 1 .

[0049] 2.2 PPI network construction

[0050] The intersection targets were imported into the STRING website to construct the PPI network, and the Cytoscape software was used for visualization analysis. The larger the node diameter and the darker the color, the higher the degree value and the more proteins interacting with it ( Figure 2 ). By calculating the Degree, Closeness, and Betweenness values, 10 core targets are obtained. See the relationship diagram for details. Figure 3 .

[0051] 2.3GO and KEGG analysis

[0052] GO functional enrichment analysis obtained a total of 689 entries based on P ≤ 0.05, including 287 biological process (BP) related entries, involving phosphorylation and protein phosphorylation, inflammatory response, etc.; 130 cell component (CC) related entries, involving plasma membrane, cytosol and cell surface, etc.; 272 molecular function (MF) related entries, involving protein tyrosine kinase activity, adenosine triphosphate binding to the same protein binding, etc. A bar graph was drawn for the top 10 BP, CC and MF analyses (see Figure 4 ).

[0053] KEGG pathway enrichment analysis obtained 155 signaling pathways according to P ≤ 0.05, including metabolic pathways, cancer pathways, neuroactive ligand-receptor interactions, etc. The top 20 pathways with relatively small P values ​​were selected to draw bubble diagrams (see Figure 5 ), the darker the color of the dot, the higher the credibility, and the larger the dot, the more genes it contains.

[0054] 2.4 Construction of “drug-ingredient-disease-pathway-target” network diagram

[0055] The "drug-ingredient-disease-pathway-target" network diagram was constructed using Cytoscape software, and 10 main therapeutic ingredients including new curcuma dione, 9-oxo-11-(3-pentyloxiran-2-YL)undec-10-enoic acid, songgolin, α-linolenic acid, atractylodes lactone III, dihydrosphingosine, Ignavine, coral atractylodesin, naked delphinium, and dihydroatisine were obtained by using De gree values ​​greater than 109.

[0056] Example 2 Pharmacodynamics study of Huafengdan on the treatment of rheumatoid arthritis

[0057] 1. Experimental Materials

[0058] 1.1 Experimental animals

[0059] 90 healthy adult female Wister rats (180-200g) were SPF grade. The breeding space was kept clean, dry and ventilated. The room temperature was controlled at 22-26°C and the humidity was 50-65%. They were given free drinking water and standard feed. The circadian rhythm was kept normal during the experiment.

[0060] 1.2 Experimental drugs and preparation

[0061] Huafengdan solution: Grind Huafengdan, weigh 1g of powder, add 10mL of distilled water, grind and mix well.

[0062] Methotrexate tablet solution: Take 1 methotrexate tablet (specification: 2.5 mg / tablet), crush it, add distilled water, grind it evenly, and prepare it into a concentration of 1 mg / kg.

[0063] 1.4 Experimental consumables

[0064] Blood collection needle, embedding box, RF kit, BAFF kit, TNF-α kit and IL-6 kit, pipette, tissue fixative

[0065] 1.5 Experimental Instruments

[0066] Automatic ice maker, electronic balance, magnetic heating stirrer, enzyme label analyzer

[0067] 2. Experimental methods

[0068] 2.1 Establishment of rheumatoid arthritis model

[0069] Preparation of immune preparation: Accurately pipette 286.0μL of 99.5% glacial acetic acid into a 50mL volumetric flask, dilute with ultrapure water, shake well, and adjust to volume to obtain 0.1mol / L acetic acid solution. Take 20mg of bovine type II collagen powder, add 0.1mol / L acetic acid solution to a 10mL volumetric flask, fully dilute and dissolve in an ice water bath, shake well after adjusting to volume, and obtain type II collagen solution. Use a small-range pipette to transfer to a sterile centrifuge tube, seal with a sealing film, and place in a 4℃ refrigerator overnight. Mix 10mL of type II collagen solution with equal volumes of pre-cooled Freund's complete adjuvant (CFA). This process is a small amount of multiple additions. Use a handheld homogenizer to fully mix and emulsify in an ice water bath. The emulsion does not disperse when it comes into contact with water, and a type II collagen emulsion with a mass concentration of 1mg / mL is obtained. The above process is always kept on an ice bath and carried out at 4℃.

[0070] Modeling method: After one week of adaptive feeding, 36 Wister rats were randomly divided into 6 groups, namely, normal group (Control group), model group (Model group), high-dose Huafengdan group (HFDH group), medium-dose Huafengdan group (HFDM group), low-dose Huafengdan group (HFDL group), and methotrexate group (MTX group), with 6 rats in each group. Except for the normal group, 0.3 mL (ensuring at least 200 μg per rat) of modeling agent emulsion was injected intradermally (avoiding blood vessels and injecting slowly) at the right hind foot and tail root of each rat. On the 7th day, a second booster immunization was performed, with each rat receiving a booster injection of 0.1 mL (ensuring that the amount of collagen per mouse was about 100 μg each time), and the blank control group was injected with an equal volume of normal saline in the same way.

[0071] 2.2 Medication and material collection

[0072] After the model was successfully established, oral administration was started, once a day, for 28 consecutive days. According to the clinical dosage, based on the body surface area of ​​humans and rats, Huafengdan (the clinical adult daily dose is 3.6g / d, which is equivalent to 0.324g / kg in rats, fully dissolved in distilled water during the experiment, once a day). Methotrexate tablets (the clinical adult daily dose is 5-10mg, 1-2 times a week; the equivalent dose of rats is 1mg / kg, fully dissolved in distilled water during the experiment, 1-2 times a week) were stored in a refrigerator at 4°C for use. The normal group and the model group were given an equal amount of solvent.

[0073] The specific dosing conditions are as follows:

[0074] Normal group: same volume of distilled water

[0075] Model group: same volume of distilled water

[0076] Positive group: methotrexate solution 1mg / kg

[0077] HFDH group: Huafengdan high-dose solution 0.648 g / kg

[0078] HFDM group: Huafengdan medium dose solution 0.324 g / kg

[0079] HFDL group: Huafengdan low-dose solution 0.162 g / kg

[0080] After 28 days of continuous administration, the rats were anesthetized with 10% ulactose intraperitoneally, and blood was collected from the abdominal aorta. The blood was left to stand at room temperature for 2 hours, and then centrifuged at 4000 r / min for 15 minutes. The supernatant was collected, packaged, and stored in a -80°C refrigerator. The synovial tissue of the rat ankle joint was also collected for use in subsequent experimental operations.

[0081] 2.3 General status observation

[0082] During the experiment, the general status changes of each group of rats were observed and recorded every day, including: activity level, mental state, hair gloss, diet, drinking water and bowel movements, plantar color, softness, swelling and refusal to press, etc.

[0083] 2.4 Body mass monitoring

[0084] During the experiment, the body weight of rats in each group was monitored every 7 days and the changes were recorded.

[0085] 2.5 Determination of foot swelling

[0086] The thickness of the plantar surface of the right hind paw of the rats was measured with a vernier caliper. The thickness was measured every 7 days during the experiment, and the degree of plantar swelling was recorded and calculated.

[0087] Plantar swelling = (C t -C0) / C0. (C0 represents the thickness of the forefoot causing inflammation, C t Indicates the thickness of the plantar fossa after inflammation.

[0088] 2.7 Hematoxylin-eosin (HE) staining of rat synovial tissue

[0089] Each group was treated separately and then HE staining was performed for pathological examination. The specific steps are as follows:

[0090] (1) After the rats were anesthetized, blood was collected from the abdominal aorta. The rats were killed, and their ankle joints were removed and fixed with 4% paraformaldehyde for later use.

[0091] (2) Decalcification of ankle joint tissue: The tissue was first fixed with 4% paraformaldehyde for 24 hours and then decalcified with EDTA. After decalcification, the joint was longitudinally sectioned, dehydrated, embedded, and sliced.

[0092] (3) Paraffin embedding: The joint tissue needs to be immersed in 50%, 70%, and 80% ethanol for 2 hours each, then immersed in 95% ethanol for 1 hour, immersed in 100% ethanol I for 1 hour, immersed in 100% ethanol II for 30 minutes, immersed in xylene I for 20 minutes, immersed in xylene II for 10 minutes, immersed in soft wax for 1 hour, immersed in hard wax for 30 minutes, and finally embedded.

[0093] (4) Dewaxing and hydration: xylene I and II for 15 min each, 100%-50% gradient ethanol for 2 min each, and rinse with tap water for 5 min.

[0094] (5) Hematoxylin staining: Stain for 8-15 minutes and wash off the hematoxylin with tap water.

[0095] (6) Differentiation: The slices were placed in 1% ethanol for 30 seconds and washed with tap water for 10 minutes.

[0096] (7) Eosin staining: stain with 1% eosin for 10 min, and wash with tap water for 10 min.

[0097] (8) Dehydration: 50%-100% gradient ethanol for 2 min each, xylene I and II for 10 min each.

[0098] (9) Sealing: Finally, use neutral gum to seal the slides.

[0099] 2.8 Micro-CT analysis

[0100] After administration, the rats were killed, and the right hind paws were immediately fixed in 4% paraformaldehyde for examination by micro-CT.

[0101] The micro-CT imaging system was used to obtain the bone tissue images of CIA rat joints. The scanning parameters were as follows: X-ray, 90 kV, 80 μA; field of view 18 mm; voxel size 36 μm; scanning mode, high resolution; scanning time 14 min. The reconstructed images after scanning were analyzed using Analysis 12.0 software.

[0102] 2.9 Elisa detection of the expression levels of inflammatory factors TNF-α, IL-1β, RF, and BAFF in rat serum. Rat serum samples were taken and the detection was performed strictly according to the operating procedures in the kit instructions.

[0103] (1) Dilution and loading of standard solution: dilute the standard solution according to the instructions (the loading volume in each well after dilution is 50 μL, and the concentrations are 240 pg / mL, 160 pg / mL, 80 pg / mL, 40 pg / mL, and 20 pg / mL, respectively);

[0104] (2) Sample addition: Pipette 10 μL of the serum sample to be tested into the sample well, then add the sample diluent to make up to 50 μL. No solution is added to the blank well. The final dilution of the sample is 5 times;

[0105] (3) Add enzyme: Add 50 mL of enzyme-labeled reagent to each well of the standard well and sample well except the blank well. Seal the reaction wells with a sealing film and incubate at 37°C in a constant temperature incubator for 30 min.

[0106] (4) Washing: discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 30 min, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times;

[0107] (5) Color development: Add 50 μL of color developing reagent A and B to each well, shake to mix, and incubate at 37°C in the dark for 10 min;

[0108] (6) Termination and measurement: Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm using an enzyme-labeled instrument.

[0109] 2.10 (Immune organ index) Determination of spleen and thymus index

[0110] After the experiment, the rats were killed, and the spleen and thymus were immediately removed, rinsed with saline, blotted dry with filter paper, weighed, and the organ index was calculated.

[0111] Immune organ index = organ mass (g) / rat body mass (g) × 100%

[0112] 2.11. Statistical methods

[0113] Statistical analysis was performed using statistical software SPSS25.0. Data followed a normal distribution and were expressed as mean ± standard deviation (X ± S). One-way ANOVA was used to compare differences between sample groups. If P < 0.05, the difference was statistically significant; if P < 0.01, the difference was statistically significant and extremely significant.

[0114] 3. Results and analysis

[0115] (1) General condition of rats

[0116] During the experiment, the model group had a depressed mental state, dull hair, reduced food and water intake, frequent licking of paws, and obvious right foot lameness when walking compared with the normal group. The drug treatment group had improved mental state, hair gloss, food and water intake, and right foot lameness when walking compared with the model group.

[0117] Before modeling, the right ankle joints of rats in each group had normal activities and no lameness. After injection of modeling drugs, all groups showed obvious redness and swelling of the right ankle joints and obvious right foot lameness. In severe cases, both ankle joints and below the ankle were red and swollen.

[0118] The swelling of the model group and the drug-treated group did not change much in the first 21 days, and the right foot was lame. However, the redness and swelling changed significantly from 21 to 28 days after drug administration, and the right foot was lame. This indicates that the acute inflammatory reaction was obvious in the early stage, and gradually turned into chronic inflammation in the later stage.

[0119] (2) Weight changes

[0120] The rats in the normal group had normal diet and activity, good growth, and steady weight gain; compared with the normal group, the rats in the model group had reduced diet, water intake, and activity, and were listless and lost weight. After intervention with Huafengdan and methotrexate, the body weight of each Huafengdan group showed an upward trend on day 21 compared with the model group, and the high-dose Huafengdan group had a significant increase in body weight, indicating that Huafengdan has a certain effect in treating rheumatoid arthritis, and the high-dose group has the best efficacy. Figure 6 .

[0121] (3) Determination of foot swelling

[0122] After the rheumatoid arthritis model was successfully established, the swelling of the rats' feet was measured once a week. The experimental results showed that the methotrexate group and the high-dose Huafengdan group had a significant reduction in swelling on the 14th day, the medium-dose group had a significant reduction on the 21st day, and the low-dose group had no significant reduction. This shows that Huafengdan has the effect of treating rheumatoid arthritis and can intervene in the development of rheumatoid arthritis. The efficacy of high-dose Huafengdan is better than that of the medium-dose and low-dose groups. See Table 2.

[0123] Table 2 The swelling of the feet of rats in each group after modeling (cm) n=6)

[0124]

[0125]

[0126] Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group, *P<0.05; **P<0.01

[0127] (5) Hematoxylin-eosin (HE) staining of rat synovial tissue

[0128] Depend on Figure 7It can be seen that in the Control group, trabeculae are abundant and regularly distributed, the bone cell structure is intact, the growth plate structure is intact, the thickness is uniform, and there is no inflammatory response; and the synovial tissue is thin and has few cells. The synovial tissue of the Model group has villous hyperplasia, that is, the synovial tissue of the joint proliferates, extending into the joint cavity in the form of villi, and there are a large number of inflammatory cells infiltrating in the villi. It can also be seen that the trabeculae are significantly reduced, the synovium proliferates, the cell layer increases, and a large number of inflammatory cells infiltrate; the chondrocytes proliferate, the number increases, the arrangement is disordered, the joint cavity becomes narrower, and cartilage destruction occurs; in the methotrexate group (MTX), after drug treatment, the conditions in the model group were reversed, and the number of trabeculae increased significantly; in the high-dose Huafengdan treatment group (HFD-H), after drug treatment, the number of trabeculae increased compared with the model group, the synovial and chondrocyte hyperplasia improved, and the joints The narrowing of the cavity has also improved; in the medium-dose treatment group of Huafengdan (HFD-M), the number of trabeculae increased compared with the model group, the proliferation of synovium and chondrocytes improved, and the narrowing of the joint cavity also improved, but the effect was worse than that of the high-dose group of Huafengdan; in the low-dose treatment group of Huafengdan (HFD-L), the effect of treating rheumatoid arthritis was not obvious after treatment, and the hyaline cartilage on the surface of the joint was destroyed and replaced by fibrous connective tissue, that is, joint fibrosis occurred. And the cartilage damage is still obvious.

[0129] (6) Micro-CT analysis

[0130] Micro-CT bone tissue related indicators

[0131] ① Number of trabeculae Tb.N (1\mm)

[0132] Trabecular bone refers to the network of calcium crystals inside the bone, which provides strength when the bone is under compression and also serves as a calcium reserve. The number of trabecular bone represents the average number of intersections between bone tissue and non-bone tissue per mm in the study area.

[0133] Results: After the rheumatoid arthritis model was successfully established, the number of trabeculae decreased, proving that bone destruction occurred. After administration, the HFD-H, HFD-M, and HFD-L groups all showed a trend of recovery, proving that Huafengdan has the effect of protecting bone destruction, among which the HFD-M group has the best protective effect. See Table 3.

[0134] Table 3 Trabecular number ( n=3)

[0135]

[0136]

[0137] Compared with the control group: #P < 0.05; ## P<0.01; compared with the model group, *P<0.05; **P<0.01

[0138] ②Bone surface area and bone volume ratio BS / TV (1\mm)

[0139] The ratio of bone surface area to bone volume is also called bone surface density, which can indirectly reflect the amount of bone mass. Rheumatoid arthritis (RA) is a chronic nonspecific inflammatory disease that mainly affects the synovial membrane of joints. Due to factors such as inflammatory response and taking glucocorticoids, patients are prone to osteoporosis (OP). Studies have shown that the incidence of OP in RA patients is as high as about half, and the risk of brittle fractures in patients is significantly increased. Therefore, early diagnosis of OP in RA patients is very important. It can not only determine whether it is OP in time, but also provide symptomatic treatment through the diagnosis results. Early detection and early treatment will make it easier to control the disease.

[0140] From a clinical perspective, establishing a correct treatment plan in a timely manner, alleviating pain, improving joint function and bone density, and preventing long-term complications are an important part of the treatment and prognosis of rheumatoid arthritis.

[0141] Results: The bone density of the rheumatoid arthritis model group was lower than that of the control group (P<0.05), which proved that the model was successfully established. After drug treatment, the bone density of the HFD-H, HFD-M, and HFD-L groups increased relative to the model group (P<0.05) (see Table 4).

[0142] Table 4 Bone surface area to bone volume ratio ( n=3)

[0143]

[0144] Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group, *P<0.05; **P<0.01

[0145] ③ Bone volume fraction BV / TV (%)

[0146] It represents the ratio of bone tissue volume to tissue volume, and its unit is %. It is a commonly used indicator for evaluating cortical bone and cancellous bone mass. It can directly reflect changes in bone mass and predict the risk of related diseases. Reduced bone mass can easily lead to osteoporosis.

[0147] Results: The bone volume fraction of the model group was lower than that of the control group (P<0.05), which proved that the model was successfully established. After drug treatment, the bone volume fraction of the HFD-H, HFD-M, and HFD-L groups increased relative to the model group (P<0.05) (see Table 5).

[0148] Table 5 Bone volume fraction ( n=3)

[0149]

[0150] Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group, *P<0.05; **P<0.01

[0151] ④ Trabecular thickness Tb.Th (mm)

[0152] The average thickness of trabeculae is used to evaluate the spatial morphology and structure of trabeculae. The object is trabeculae, and the value decreases when osteoporosis occurs.

[0153] Results: The trabecular thickness of the model group was significantly lower than that of the normal group (P<0.05), which proved that the model was successfully established. After drug treatment, the trabecular thickness of the HFD-H, HFD-M, and HFD-L groups increased relative to the model group (P<0.05), as shown in Table 6.

[0154] Table 6 Trabecular thickness ( n=3)

[0155]

[0156] Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group, *P<0.05; **P<0.01

[0157] (7) Elisa detection of the expression levels of TNF-α, IL-1β (key cytokines in CIA rats), RF, and BAFF inflammatory factors in rat serum

[0158] RF is a serological index determined by the diagnostic criteria for rheumatoid arthritis, and is of great significance for the diagnosis, typing and efficacy observation of rheumatoid arthritis. After treatment, the RF content in the serum of rats was significantly reduced, showing the clinical advantages of the drug.

[0159] BAFF can specifically activate B lymphocytes, and abnormal overexpression of BAFF can cause autoimmune diseases (Bosello et al, 2007). The results showed that BAFF was highly expressed in the serum of mice in the model group. After HFD treatment, the expression level of BAFF was significantly inhibited. The abnormal immune activation of mice was inhibited and the symptoms of RA were alleviated.

[0160] Rheumatoid factor (RF) is the earliest serological indicator used for the diagnosis of RA and the only serological indicator proposed by the American College of Rheumatology (ACR) in the diagnostic criteria for RA (Sobhy et al, 2022). B cell activating factor (BAFF) belongs to the tumor necrosis factor superfamily and is closely related to the occurrence and development of autoimmune diseases.

[0161] Table 7 Expression levels of RF and BAFF in serum under different groups

[0162]

[0163] Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group, *P<0.05; **P<0.01

[0164] The expression levels of RF and BAFF in serum are shown in Table 7. The expression levels of RF and BAFF in the model group were significantly higher than those in the control group (p<0.001), indicating that the CIA model was successfully established. After HFD treatment, the serum RF and BAFF levels were significantly reduced (p<0.01). In summary, HFD can inhibit abnormal immune responses and relieve RA symptoms, among which the high-dose group of Huafengdan has the best efficacy. (See Figures 8 to 9 )

[0165] RA is a systemic chronic disease characterized by synovitis. Middle-aged women are the most susceptible group, and the incidence rate in my country is about 0.35%. Most studies have shown that the pathogenesis of RA is still unclear, but it is related to changes in genes, environment and other factors, and patients experience destructive joint inflammation. A large number of studies have shown that synovial cells play a key role in maintaining joint balance. The synovial cells of RA patients are more active and can secrete a variety of inflammatory factors, damage cartilage, and worsen the condition. TNF-α is a proinflammatory cytokine. Studies have shown that TNF-α can induce other cytokines to participate in the RA inflammatory response by activating multiple signals, and plays a key role in the occurrence and development of RA diseases. Interleukin 1β, as a proinflammatory agent, can activate inflammatory responses and induce rheumatoid arthritis.

[0166] Table 8 Expression levels of TNF-α and IL-1β in serum under different groups

[0167]

[0168]

[0169] Compared with the control group: # P < 0.05;## P<0.01; compared with the model group, *P<0.05; **P<0.01.

[0170] The expression levels of TNF-α and IL-1β in serum are shown in Table 8. The expression levels of TNF-α and IL-1β in the model group were significantly higher than those in the control group (p<0.001), indicating that the CIA model was successfully established. After HFD treatment, the serum TNF-α and IL-1β levels were significantly reduced (p<0.01). In summary, HFD can inhibit inflammatory response and relieve RA symptoms, among which the high-dose group of Huafengdan has the best efficacy. (See Figure 10 to Figure 11 )

[0171] (8) Immune organ index

[0172] The thymus and spleen are important lymphatic organs, and abnormalities of these organs are closely related to the occurrence of immune system diseases.

[0173] Table 9 Immune organ index ( n=6)

[0174]

[0175] Compared with the control group: # P < 0.05; ## P<0.01; compared with the model group, *P<0.05; **P<0.01

[0176] The experimental results show that (see Table 9) the immune organ tissues in the model group were significantly higher than those in the blank group, indicating that the rheumatoid arthritis model was successfully established. After drug treatment, the thymus index of the high-dose Huafengdan group had a significant correction, and the spleen index of the high-dose and medium-dose Huafengdan groups decreased. This proves that the high-dose and medium-dose Huafengdan groups can treat rheumatoid arthritis.

[0177] The embodiments described in the present invention are merely descriptions of the preferred implementation modes of the present invention, and are not intended to limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various modifications and improvements made to the technical solutions of the present invention by those skilled in the art should all fall within the protection scope of the present invention.

Claims

1. A new use of Huafengdan, the formula of which is composed of 45.5 parts of mother medicine, 40.725 parts of perilla leaves, 18.2 parts of bombyx batryticatus, 9.175 parts of scorpion, 9.175 parts of processed arisaema, 9.175 parts of atractylodes, 6.675 parts of realgar, 18.125 parts of borax, 3.125 parts of croton frost, 0.625 parts of artificial musk, 9.175 parts of borneol, 18.2 parts of gastrodia, 4.575 parts of schizonepeta, 0.825 parts of sandalwood, and 6.05 parts of cinnabar, and the mother medicine is composed of 126.7 parts of ox bile, 28.2 parts of white aconite, 28.2 parts of raw pinellia, 28.2 parts of raw arisaema, 28.2 parts of raw Chuanwu, 14.1 parts of curcuma, and 0.14 parts of Shenqu, characterized in that The new use is the use of Huafengdan in the preparation of medicines for treating rheumatoid arthritis.

2. An analytical method for the new use of Huafengdan as claimed in claim 1, characterized in that: The analysis method is to use network pharmacology and molecular docking technology to analyze the mechanism of action of Huafengdan in treating rheumatoid arthritis, including five steps: screening of active ingredients and potential targets, screening of diseases and intersection targets, construction of PPI network and core target screening, GO enrichment analysis and KEGG pathway analysis, and construction of "drug-active ingredient-disease-pathway-target" network.

3. The analysis method according to claim 2, characterized in that The screening of active ingredients and potential targets is specifically as follows: the active ingredients of Huafengdan are screened using the PubChem database and the Swiss ADME website, gastrointestinal absorption is high, and at least two of the five drug-like principles are met if the results are "Yes", and finally the ingredient targets are queried using the SwissTargetPredictio website.

4. The analysis method according to claim 2, characterized in that The screening of the disease and intersection targets is specifically as follows: using "rheumatoid arthritis" as a keyword, collecting disease targets in the OMIM, TTD and Gene Cards databases, deleting duplicates to obtain disease targets, and drawing a VENNY diagram to obtain component and disease intersection targets.

5. The analysis method according to claim 2, characterized in that The construction of the PPI network and the screening of core targets are specifically as follows: the intersection targets are imported into the STRING website to construct a PPI network diagram, and the Cytoscape software is used to perform a visualization analysis on it, and then the "CytoNCA" plug-in is used to perform a topological analysis, and the core targets are selected using the degrees of freedom, closeness centrality and betweenness centrality as quantitative indicators.

6. The analysis method according to claim 2, characterized in that The GO enrichment analysis and KEGG pathway analysis are specifically as follows: the intersection targets are imported into the DAVID database, GO function analysis and KEGG pathway enrichment analysis are performed, and then the top results are selected according to the P value sorting and visualized on the microbial information platform.

7. The analysis method according to claim 2, characterized in that The "drug-active ingredient-disease-pathway-target" network construction is specifically as follows: using Cytoscape software to construct a "drug-active ingredient-disease-pathway-target" network diagram, and selecting components with Degree values ​​≥ 109 as the main therapeutic components.

8. The analysis method according to claim 7, characterized in that The main therapeutic ingredients are new curcuma dione, 9-oxo-11-(3-pentyloxiran-2-YL)undec-10-enoic acid, songguoling, α-linolenic acid, atractylodes lactone III, dihydrosphingosine, Ignavine, coral atractylodesin, naked delphinium, and dihydroatisine.

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