Application of ointment capable of dredging collaterals and relieving pain in treatment effect of pain

Abstract

The invention discloses application of ointment for dredging collaterals and relieving pain in the treatment effect of pain, and belongs to the field of biomedicine. The technical problem to be solved by the invention is how to detect the treatment effect of the collateral-dredging and pain-relieving ointment on pain. The compounds disclosed by the invention are fourteen compounds of which the contents are obviously increased in a rabbit pain model and the contents are obviously reduced after treatment by the collateral-dredging and pain-relieving paste, and the treatment effect of the collateral-dredging and pain-relieving paste is detected by utilizing each compound, so that the accuracy is good. The fourteen compounds provided by the invention not only can be used for detecting the treatment effect of the ointment for dredging collaterals and relieving pain, but also can be used for further screening individuals with skeletal pain, and also can be used for preparing corresponding animal models by utilizing the ointment for dredging collaterals and relieving pain, thereby providing a basis for further researching which compounds participate in skeletal pain. And a new therapeutic target can be provided for the treatment of bone pain.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and particularly relates to the application of Tongluo Qutong ointment in the treatment of pain. Background Art

[0002] Tongluo Qutong Paste is a topical medication primarily composed of Chinese medicinal herbs such as Angelica sinensis and Chuanxiong rhizome. It primarily promotes blood circulation and dredges the meridians, dispels cold and dampness, and relieves swelling and pain. It is indicated for lumbar and knee osteoarthritis characterized by blood stasis and cold-dampness blocking the meridians. Symptoms include stabbing or dull pain in the joints, stiffness, difficulty flexing and extending, and chills and cold extremities. However, the active molecule in Tongluo Qutong Paste's treatment of chronic pain remains unknown, necessitating the testing of its effectiveness in treating pain. Summary of the Invention

[0003] The first technical problem to be solved by the present invention is how to prepare an animal model.

[0004] The present invention first provides a method for preparing an animal model for non-therapeutic purposes (denoted as Method 1), wherein Method 1 comprises applying Tongluo Qutong ointment to the painful area of a painful individual, thereby obtaining an animal model having at least one of the following characteristics:

[0005] D1) Histamine levels decreased;

[0006] D2) the content of trimethylamine oxide decreased;

[0007] D3) Gossypol content decreased;

[0008] D4) 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid levels decreased;

[0009] D5) 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone content decreased;

[0010] D6) 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine content decreased;

[0011] D7) 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine content decreased;

[0012] D8) 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide content decreased;

[0013] D9) Benzoic acid,2,4-dimethoxy-6-methyl-,4-carboxy-3-methoxy-5-methylphenyl ester content decreased;

[0014] D10) Betazole content decreased;

[0015] D11) Kipukasin A content decreased;

[0016] D12) Piperazine content decreased;

[0017] D13) Thiazovivin levels decreased;

[0018] D14) Warfarin content decreased.

[0019] In one embodiment of the present invention, the Tongluo Qutong ointment is a high-dose Tongluo Qutong ointment, a medium-dose Tongluo Qutong ointment or a low-dose Tongluo Qutong ointment.

[0020] Among them, the high-dose Tongluo Qutong ointment can be obtained by preparing the Tongluo Qutong ointment raw material at 0.9 g / kg body weight.

[0021] The medium-dose Tongluo Qutong ointment can be prepared by adding 0.3 g / kg body weight of the Tongluo Qutong ointment crude drug to the Tongluo Qutong ointment.

[0022] The low-dose Tongluo Qutong ointment can be prepared by adding 0.1 g / kg body weight of the Tongluo Qutong ointment crude drug to the Tongluo Qutong ointment.

[0023] Among them, the raw materials of Tongluo Qutong Paste can be prepared using the following Chinese medicinal materials in parts by mass: 110g of angelica, 66g of Chuanxiong, 68g of safflower, 52g of galangal, 72g of Zanthoxylum, 72g of pepper, 36g of cloves, 45g of cinnamon, 52g of piper longum, 56g of dried ginger, 56g of rhubarb, 46g of camphor, 31g of borneol, and 31g of menthol.

[0024] In the above method 1, the pain-suffering individual may be a pain-suffering individual of a mammal (such as a rabbit).

[0025] In one embodiment of the present invention, the Tongluo Qutong ointment may be the above-mentioned high-dose Tongluo Qutong ointment.

[0026] The present invention also provides another method for preparing an animal model for non-therapeutic purposes (denoted as Method 2), wherein Method 2 comprises: injecting iodoacetic acid into an animal to obtain an animal model having at least one of the following characteristics:

[0027] D1) Increased histamine levels;

[0028] D2) increased trimethylamine oxide levels;

[0029] D3) increased gossypol content;

[0030] D4) increased levels of 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid;

[0031] D5) increased levels of 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone;

[0032] D6) 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine content increased;

[0033] D7) increased levels of 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine;

[0034] D8) increased levels of 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide;

[0035] D9) Benzoic acid,2,4-dimethoxy-6-methyl-,4-carboxy-3-methoxy-5-methylphenyl ester content increased;

[0036] D10) Betazole levels increased;

[0037] D11) Kipukasin A content increased;

[0038] D12) Piperazine levels increased;

[0039] D13) Thiazovivin levels increased;

[0040] D14) Warfarin levels increased.

[0041] In the above method 2, injecting iodoacetic acid into the animal may comprise injecting iodoacetic acid into the knee joint cavity (such as the knee joint cavity of the leg) of the animal.

[0042] In the above method 2, the injection amount of iodoacetic acid can be 0.012 mg iodoacetic acid per gram of body weight.

[0043] In the above-mentioned methods 1 and 2, the content of histamine, trimethylamine oxide, gossypol, 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylicacid, 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone, 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine, 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine, 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide, Benzoicacid, 2,4-dimethoxy-6-methyl-, 4-carboxy-3-methoxy-5-methylphenyl ester, Betazole, Kipukasin A, Piperazine, Thiazovivin or Warfarin may be the content in joint tissue.

[0044] In the above method 1 and method 2, the animal may be a mammal (such as a rabbit).

[0045] In the above method 1 and method 2, the animal model can be a rabbit model.

[0046] The second technical problem to be solved by the present invention is how to detect the therapeutic effect of Tongluo Qutong Paste on pain, and how to screen individuals with pain.

[0047] To solve the above technical problems, the present invention first provides at least one of the following applications of a substance for detecting compound content:

[0048] X1. Use in the preparation of a product for detecting or assisting in detecting the therapeutic effect of Tongluo Qutong Cream on pain, or in detecting or assisting in detecting the therapeutic effect of Tongluo Qutong Cream on pain;

[0049] X2. Use in the preparation of a product for screening or assisting in screening individuals experiencing pain after treatment with Tongluo Qutong Cream, or use in screening or assisting in screening individuals experiencing pain after treatment with Tongluo Qutong Cream;

[0050] X3. Use in the preparation of products for screening or assisting in the screening of individuals with pain, or in the screening or assisting in the screening of individuals with pain;

[0051] The compounds are histamine, trimethylamine oxide, gossypol, 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid, 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone, 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine , 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine , 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide , Benzoic acid,2,4-dimethoxy-6-methyl-,4-carboxy-3-methoxy-5-methylphenyl ester, Betazole, Kipukasin A, Piperazine, Thiazovivin or / and Warfarin.

[0052] Among them, the CAS number of histamine is 51-45-6;

[0053] The CAS number of trimethylamine oxide is 1184-78-7;

[0054] The CAS number of gossypol is 90141-22-3;

[0055] The CAS number of 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid is 187998-44-3;

[0056] The CAS number of 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone is 4644-03-5;

[0057] The CAS number of 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine is 26662-95-3;

[0058] The CAS number of 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine is 4875-41-6;

[0059] The CAS number of 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide is 1717-36-8;

[0060] The CAS number of Benzoic acid,2,4-dimethoxy-6-methyl-,4-carboxy-3-methoxy-5-methylphenyl ester is 913691-43-7;

[0061] The CAS number of Betazole is 105-20-4;

[0062] The CAS number of Kipukasin A is 948573-97-5;

[0063] The CAS number of Piperazine is 110-85-0;

[0064] The CAS number of thiazovivin is 1226056-71-8;

[0065] The CAS number of warfarin is 81-81-2.

[0066] In the above application, the pain may be bone pain.

[0067] In one embodiment of the present invention, the bone pain is bone pain in an animal model obtained by injecting iodoacetic acid into the knee joint cavity at a rate of 0.012 mg iodoacetic acid per gram of body weight.

[0068] In one embodiment of the present invention, the animal model is a rabbit model.

[0069] In the above application, the content of the compound is the content of the compound in the joint tissue of an animal individual (such as a rabbit).

[0070] In the above application, the substance for detecting the compound content may also contain a data conversion device, which is used to convert the numerical value of the compound content into the therapeutic effect value of the Tongluo Qutong Paste on pain or the pain value of the individual with pain.

[0071] In the above applications, the substance for detecting the compound content can be a substance capable of detecting the compound content, such as a substance (such as a reagent, etc.) used to detect the compound content using an LC-MS method. The substance for detecting the compound content can include a standard of the compound (i.e., the compound itself).

[0072] In one embodiment of the present invention, the Tongluo Qutong ointment is a high-dose Tongluo Qutong ointment, a medium-dose Tongluo Qutong ointment or a low-dose Tongluo Qutong ointment.

[0073] Among them, the high-dose Tongluo Qutong ointment is obtained by preparing the Tongluo Qutong ointment by mixing the raw materials of Tongluo Qutong ointment at 0.9 g / kg body weight.

[0074] The medium-dose Tongluo Qutong ointment is prepared by adding 0.3 g / kg body weight of the Tongluo Qutong ointment crude drug to the Tongluo Qutong ointment.

[0075] The low-dose Tongluo Qutong ointment is prepared by adding 0.1 g / kg body weight of the Tongluo Qutong ointment crude drug to the Tongluo Qutong ointment.

[0076] Among them, the raw materials of Tongluo Qutong Paste are prepared using the following Chinese medicinal materials in parts by mass: 110g of angelica, 66g of Chuanxiong, 68g of safflower, 52g of galangal, 72g of Zanthoxylum, 72g of pepper, 36g of cloves, 45g of cinnamon, 52g of piper longum, 56g of dried ginger, 56g of rhubarb, 46g of camphor, 31g of borneol, and 31g of menthol.

[0077] In the above application, the therapeutic effect of the Tongluo Qutong ointment on pain can be the therapeutic effect of the Tongluo Qutong ointment on pain in humans or animals (such as rabbits).

[0078] The subject in pain may be a human or an animal (eg, rabbit) subject in pain.

[0079] In the present invention, the bone pain may be chronic bone pain.

[0080] In the present invention, the therapeutic effect can be reflected in the expression level of inflammatory factors. The inflammatory factors can be IL-6, IL-1β and / or TNF-α. The inflammatory factors can be inflammatory factors in the joint area.

[0081] In one embodiment of the present invention, the therapeutic effect is that the expression levels of IL-6 and IL-1β after treatment are reduced to 1 / 2 of that before treatment, and TNF-α after treatment is reduced to 2 / 5 of that before treatment.

[0082] In the present invention, the therapeutic effect may also be reflected in the paw withdrawal threshold and / or the paw withdrawal delay time.

[0083] The invention uses Tongluo Qutong ointment to treat rabbit bone pain and finds that in the rabbit pain model, histamine, trimethylamine oxide, gossypol, 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid, 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone, 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine, 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine, 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide, Benzoic acid, 2,4-dimethoxy-6-methyl-, 4-carboxy-3-methoxy-5-methylphenylester, Betazole, Kipukasin The contents of fourteen compounds, including A, Piperazine, Thiazovivin and Warfarin, increased significantly, while after treatment with Tongluo Qutong Ointment, the contents of these compounds decreased significantly, indicating that a basis can be provided for further studying which compounds are involved in bone pain by preparing corresponding animal models, and that new therapeutic targets can be provided for the treatment of bone pain. Detecting the therapeutic effect of Tongluo Qutong Ointment using the fourteen compounds of the present invention has good accuracy, and can not only be used to detect the therapeutic effect of Tongluo Qutong Ointment, but also can be used to further screen individuals with bone pain. In practical applications, detecting the therapeutic effect of Tongluo Qutong Ointment on rabbit bone pain, or preparing animal models with changes in the contents of the fourteen compounds, can not only lay the foundation for further clinically detecting the therapeutic effect of Tongluo Qutong Ointment on human bone pain, but can also be used to study which of these compounds and their upstream and downstream compounds are involved in bone pain, and provide new therapeutic targets for further treatment.

[0084] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 .Correlation analysis of differentially expressed genes and metabolites (model group vs. injection control group).

[0086] Figure 2.Correlation analysis of differentially expressed genes and differentially expressed metabolites (model group vs. Tongluo Qutong ointment high-dose group). DETAILED DESCRIPTION

[0087] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, and instruments used in the following examples are all commercially available unless otherwise specified.

[0088] The quantitative experiments in the following examples were all repeated at least three times, and the data were processed using SPSS 11.5 statistical software. The experimental results were expressed as mean ± standard deviation, and the significance analysis was performed using One-way ANOVA test.

[0089] Example 1

[0090] 1. Animal Experiment on Chronic Bone Pain

[0091] In order to test the therapeutic effect of Tongluo Qutong Ointment at the animal level, the inventors constructed a rabbit model of chronic bone pain. The specific construction steps are as follows:

[0092] Rabbits weighing approximately 3.0±0.12 kg were randomly divided into seven groups: normal group, injection control group, model group, positive drug group, and Tongluo Qutong ointment high-, medium-, and low-dose groups, with 15 rabbits in each group. After three days of adaptive training, the rabbits in each group were treated as follows:

[0093] Normal group: no treatment was performed and served as control;

[0094] Injection control group (i.e., sham operation group): On the first day, an injection was made into the rabbit's knee joint cavity, and the needle was removed without any treatment;

[0095] Model group: On the first day, 3 mg of iodoacetic acid (i.e., 0.012 mg of iodoacetic acid per gram of body weight) was injected into the knee joint cavity of the rabbit's hind leg to establish a rabbit model of bone pain;

[0096] Positive drug group: One hour after successful model establishment, dexamethasone injection (2 mg / kg) was intraperitoneally injected. Starting from the second day, the drug was administered once a day and the treatment continued until the seventh day.

[0097] Tongluo Qutong Ointment High-Dose Group: One hour after successful model establishment, high-dose Tongluo Qutong Ointment was applied to the knee joint. Treatment began on the second day and continued until the seventh day, with the ointment applied once daily for six hours each time.

[0098] Medium-dose Tongluo Qutong Ointment group: One hour after successful model establishment, medium-dose Tongluo Qutong Ointment was applied to the knee joint. Treatment began on the second day and continued until the seventh day, with the ointment applied once daily for 6 hours each time.

[0099] Tongluo Qutong Ointment Low-Dose Group: One hour after successful model establishment, low-dose Tongluo Qutong Ointment was applied to the knee joint. Treatment began on the second day and continued until the seventh day, with the ointment applied once daily for six hours each time.

[0100] Among them, the preparation of high-dose Tongluo Qutong ointment: Tongluo Qutong ointment crude drug is prepared at 0.9g / kg body weight, which is equivalent to 9 times the clinical dose of humans;

[0101] Preparation of medium-dose Tongluo Qutong ointment: 0.3 g / kg body weight of the raw material of Tongluo Qutong ointment was used to prepare Tongluo Qutong ointment, which is equivalent to 3 times the clinical dose for humans.

[0102] Preparation of low-dose Tongluo Qutong ointment: 0.1 g / kg body weight of the Tongluo Qutong ointment crude drug was used to prepare the Tongluo Qutong ointment, which is equivalent to the human clinical dose.

[0103] Among them, the raw materials of Tongluo Qutong Paste are prepared using the following Chinese medicinal materials in parts by weight: 110g of angelica, 66g of Chuanxiong, 68g of safflower, 52g of galangal, 72g of Zanthoxylum, 72g of pepper, 36g of cloves, 45g of cinnamon, 52g of longan, 56g of dried ginger, 56g of rhubarb, 46g of camphor, 31g of borneol, and 31g of menthol. Its production method is as follows:

[0104] First, 110g of angelica, 66g of chuanxiong, 68g of safflower, 52g of kaempferia, 72g of zanthoxylum, 56g of dried ginger, 72g of pepper, 36g of cloves, 56g of rhubarb, and 52g of longan were crushed into coarse powder (they can be crushed separately and then mixed, or crushed together), and the extract and the percolation method under the extract (Appendix IO of the 1995 edition of the Chinese Pharmacopoeia) were used. After soaking in 90% ethanol for 24 hours, the percolation was collected. 1100ml of the percolation liquid was collected, and the residue was then added with solvent gasoline for extraction 3 times, with the amount of gasoline added being 420ml for the first time, 220ml for the second and third times, to obtain the extracted oil. After 45g of cinnamon bark was crushed separately, it was soaked in 90% ethanol for 24 hours and percolated. 120ml of the percolation liquid was collected and extracted 3 times with gasoline, with 45ml of gasoline added for the first time, and 25ml for the second and third times, to obtain the extracted oil. The oils were mixed together in a volume ratio of 9:1 and set aside; the two medicinal residues after oil extraction were respectively percolated with 70% ethanol by mass concentration, and 2300 ml of the percolate of angelica, Chuanxiong, safflower, galangal, Zanthoxylum, dried ginger, pepper, clove, longan and rhubarb, and 260 ml of the percolate of cinnamon were collected. The two percolate solutions were combined (volume ratio of 9:1), and concentrated under reduced pressure to a relative density of not less than 1 at 80°C. .20. The extract with a water content not exceeding 18.0% is set aside; take 290g of rubber paste, 270g of rosin, 360g of zinc oxide, 30g of vaseline, 70g of lanolin, 20g of liquid paraffin, 12g of dimethyl sulfoxide, and 1300ml of gasoline to make a matrix to eliminate static electricity, then add the above extract, extracted oil and the above camphor, borneol and menthol, mix evenly, apply on the base cloth, cover with a patch layer, and slice to make the required meridian-clearing and pain-relieving ointment.

[0105] Von Frey Hair test and Hot Plate test (hot plate experiment) were performed on the 6th, 10th and 14th days respectively. The results of Von Frey Hair test showed that during the experimental period, the paw withdrawal thresholds of the low, medium and high dose groups of Tongluo Pain Relief Cream were significantly higher than those of the model group. Among them, the therapeutic effects of the high dose group of Tongluo Pain Relief Cream and the dexamethasone (positive drug group) were close, and the rabbits had the mildest symptoms, indicating that the high dose group of Tongluo Pain Relief Cream had a good therapeutic effect. The Hot Plate test experiment was used to evaluate the effect of the drug on the pain threshold of rabbits. The results showed that during the experimental period, the paw withdrawal delay time of the Tongluo Pain Relief Cream treatment group was significantly better than that of the model group, and the therapeutic effect of the high dose group of Tongluo Pain Relief Cream was similar to that of the positive drug group. The above results prove that Tongluo Pain Relief Cream has a good alleviating effect on bone damage occurring in the process of chronic bone pain.

[0106] Inflammatory factors are one of the key factors causing pain. To verify the efficacy of Tongluo Pain Relief Cream in alleviating chronic bone pain, the inventors tested the expression of inflammatory factors in the rabbit joints after treatment on the 7th day. Gene expression results showed that compared with the model group, the secretion of inflammatory factors in the high-dose Tongluo Pain Relief Cream group was significantly reduced, with IL-6 and IL-1β reduced to 1 / 2 and TNF-α reduced to 2 / 5 of the model group, both of which were consistent with the control group, demonstrating that the high-dose Tongluo Pain Relief Cream group effectively reduced the inflammatory response in the rabbit joints.

[0107] 2. Transcriptomics detection of Tongluo Qutong ointment in the treatment of chronic pain

[0108] 2.1 Transcriptomics results

[0109] To further elucidate the molecular mechanism of Tongluo Qutong ointment in treating chronic bone pain, the inventors conducted transcriptomic testing on the rabbit joint tissues after administration on the 7th day. The screening criteria for differentially expressed genes were log2FoldChange>1 and pvalue<0.05.

[0110] Notably, the expression of the pain-related gene Trpv1 was significantly suppressed after drug administration. As a membrane-channel receptor closely associated with pain transmission, TRPV1 plays an important role in mediating various pain pathways, including inflammatory pain, visceral pain, cancer pain, and even pain sensitization. Furthermore, the expression of the anti-inflammatory gene Acod1 increased after drug administration. Acod1 encodes a mitochondrial enzyme that produces itaconate and exerts anti-inflammatory effects by promoting Nrf2 expression following lipopolysaccharide induction. Furthermore, Ebi3 gene expression was induced after drug administration. Ebi3 is a β subunit of the IL-12 cytokine family and typically binds to α subunits p19, p28, or p35 to form heterodimeric cytokines IL-39, IL-27, and IL-35, respectively. Proteins that bind to Ebi3 include IL-6 and other IL-12 family β subunits, p40. Ebi3 also has the ability to bind to various cytokines with varying structural similarities to the IL-12 family α subunit. Ebi3 can also bind to the pro-inflammatory factor IFN-γ and eliminate the signal transduction and function of its downstream genes, ultimately exerting an anti-inflammatory effect.

[0111] It can be seen that Tongluo Qutong Paste has a wide range of molecular networks regulated at the molecular level. It can both relieve pain and reduce inflammation, and has a good therapeutic effect on chronic bone pain.

[0112] 2.2 Analysis of enriched pathways of differentially expressed genes

[0113] The GO database was searched for signal pathway enrichment of differentially expressed genes. GO enrichment analysis showed that, based on the GO enrichment of differentially expressed genes that were elevated in the model group and decreased after drug administration, most genes were enriched in the following cellular components: immune synapse, intercellular junction, T cell receptor complex, cell surface, and plasma membrane; and in the following cellular processes: cell surface receptor signaling pathway, calcium-mediated signaling, positive regulation of integrin-mediated intercellular adhesion, immune response, T cell receptor signaling pathway, cellular response to type II interferon, chemokine-mediated signaling pathway, T cell proliferation, positive regulation of leukocyte adhesion, and cell chemotaxis; and in the following molecular functions: transmembrane signaling receptor activity, phospholipase activator activity, CC chemokine binding, and CC chemokine receptor activity. As can be seen, most of these genes are related to immunity and inflammation.

[0114] According to the GO enrichment of differentially expressed genes that were decreased in the model group and increased after drug administration, it was found that most genes were enriched in the following cellular components: myosin filaments, myosin II complex, myosin complex, muscle myosin complex, plasma membrane, and myofibril; enriched in the following cellular processes: myofilament sliding, ATP metabolism process, sarcomere organization, embryonic skeletal system morphogenesis, and cartilage development; and the following molecular functions: microfilament movement activity, calmodulin binding, actin filament binding, ATP binding, extracellular ligand-gated single-atom ion channel activity, and cytoskeleton movement activity.

[0115] Further, the inventors carried out KEGG pathway enrichment analysis by KEGG database for differentially expressed genes. According to the model group, the genes and signaling pathways of the differentially expressed genes that decrease after administration are enriched, it can be seen that most of the genes are enriched in Th17 cell differentiation, cytokine-cytokine receptor interaction and Th1 and Th2 cell differentiation signaling pathways, and these pathways are closely related to immune and inflammatory pathological processes, and are also closely related to pain. On the other hand, according to the model group, the genes and signaling pathways of the differentially expressed genes that increase after administration are enriched, it can be seen that most of the genes are enriched in motor proteins and cytoskeleton in muscle cells, and these genes are closely related to processes such as the cytoskeleton in exercise, metabolism, and muscle cells. The above results show that Tongluo Qutong Paste improves the function of cytoskeleton and motor protein in muscle cells around joint tissue by reducing inflammation, regulates metabolite levels, and relieves bone pain.

[0116] 3. Metabolomics testing of Tongluo Qutong ointment for the treatment of chronic pain

[0117] 3.1 Metabolomics results

[0118] The perception of physical pain is closely related to changes in the content of metabolites in the body. The inventors further conducted metabolomics testing on the hind leg joint tissues of rabbits after treatment with Tongluo Qutong ointment and analyzed the differential metabolites. The steps of metabolomics testing are as follows: (1) experimental testing; (2) data analysis. Among them, the experimental part includes blood sample collection, metabolite extraction, and metabolite detection using LC-MS. The data analysis includes raw data processing, data quality control, data evaluation, and statistical analysis. Statistical analysis uses univariate statistical analysis and multivariate statistical analysis methods to screen out metabolites with differences between groups, and then analyze their metabolic pathways.

[0119] By comparing the differential metabolites between the high-dose Tongluo Pain Relief Paste group and the model group, and the intersection of the differential metabolites between the high-dose Tongluo Pain Relief Paste group and the model group, a total of 15 metabolites were found to exist in both groups (Table 1).

[0120] Table 1. List of differential metabolites

[0121]

[0122] 3.2 Correlation Analysis Hierarchical Cluster Analysis

[0123] The inventors performed a correlation hierarchical cluster analysis on the differential metabolites and screened out 14 metabolites that were significantly downregulated in the injection control group and the Tongluo Qutong ointment high-dose group compared with the model group from 15 differential metabolites: 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid; 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone; 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine; 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine; 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide; Benzoic acid, 2,4-dimethoxy-6-methyl-, 4-carboxy-3-methoxy-5-methylphenyl ester; Betazole; Gossypol; Histamine; Kipukasin A; Piperazine; Thiazovivin; Trimethylamine N-oxide; Warfarin.

[0124] 3.3Box-plot analysis of differential metabolite expression levels

[0125] The inventors calculated the significance p-value for the quantitative values of the 14 differential metabolites screened out in 3.2 and found that among these metabolites, the increased levels of 3 metabolites were closely related to pain, while the levels of these metabolites were significantly decreased after treatment with Tongluo Pain-Relieving Ointment, namely histamine, trimethylamine oxide and gossypol.

[0126] (1) Histamine: Histamine is an organic nitrogen compound produced by histidine under the action of decarboxylase. Many tissues, especially mast cells in the skin, lungs and intestinal mucosa, contain large amounts of histamine. Histamine can be released when tissues are damaged or inflammation and allergic reactions occur. Histamine has a strong vasodilator effect and can increase the permeability of the walls of capillaries and venules, allowing plasma to leak into tissues, leading to local tissue edema. Histamine acts through four different GPCR subtypes: H1, H2, H3 and H4 receptors. Excitatory histamine receptor signals in the pathway are associated with increased pain symptoms. The H1 receptor is an excitatory receptor that couples to Gq-type proteins, leading to downstream activation of PLC and phosphatidylinositol 4,5-bisphosphate (PIP2) to produce DAG and inositol triphosphate (IP3). DAG then activates PKC on the membrane, while IP3 diffuses and binds to IP3 receptors on the endoplasmic reticulum to mobilize stored calcium. These changes lead to depletion of PIP2 calcium and intracellular Ca 2+ Increased concentrations activate PKC-dependent phosphorylation and form a complex with calmodulin (Ca 2+ CaM), both of which inhibit potassium voltage-gated type 7 channels, leading to depolarization and increased nociception (Brown & Passmore, 2009; Chen, Li, Hiett, & Obukhov, 2016). Therefore, the inventors speculate that Tongluo Qutong Paste can further alleviate chronic pain symptoms by inhibiting histamine production, thereby reducing the activation of H1 receptor signaling.

[0127] (2) Trimethylamine N-oxide (TMAO): Molecules with a molecular weight of 75.11 and less than 10 kDa are considered to be easily absorbed through synovial capillaries. Intra-articular injection of TMAO revealed that IL6 levels in rabbits treated with TMAO and anterior cruciate ligament transection surgery were significantly increased. IL6 is an important characteristic factor of knee arthritis, and the increase in IL-6 exacerbated the pain in rabbits. NF-κB is a major regulator of inflammation and bone remodeling. Increased NF-κB activity is accompanied by enhanced bone resorption and decreased bone formation capacity. Studies have shown that NF-κB p65 expression increases after TMAO treatment. Conversely, the use of NF-κB inhibitors significantly reduces the production of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and ROS release. In addition, trimethylamine N-oxide can enhance the expression of the Piezo1 gene in chondrocytes. When mechanical stress is applied, the Piezo1 channel blades move like levers, which causes the central pore to open and allow positive charges to flow in. Overexpression of the Piezo1 channel leads to intracellular Ca 2+ levels and induces "mechanical hypersensitivity," a response to mechanical strain-induced Ca 2+ Influx, Ca 2+ Influx leads to sparse F-actin cytoskeleton, reduces chondrocyte stiffness and increases cell deformation in response to mechanical load. Piezo1 channels are activated in response to excessive mechanical strain, leading to cytoskeleton disaggregation, chondrocyte metabolic disorders and cell apoptosis. In addition, high serum concentrations of TMAO are closely related to systemic and local inflammation (Coras et al., 2019; Chou et al., 2019). Based on this, the inventors believe that Tongluo Qutong Paste reduces the secretion of various inflammatory factors by reducing the content of trimethylamine oxide in rabbits, while inhibiting the opening of the mechanically sensitive ion channel protein Piezo1, reducing Ca 2+ Flowing in and thus relieving chronic pain.

[0128] (3) Gossypol: As a polyphenolic compound, gossypol interferes with cell metabolism in many ways by binding to membrane matrix and protein molecules, and possibly destroying membrane structure and interfering with the electron transport system through free radical mechanisms. It can exhibit anti-fertility, anti-inflammatory, anti-tumor, anti-vasodilator, anti-viral, anti-parasitic activities, as well as various toxicities. Cottonseed meal and cottonseed protein concentrate are protein substitutes for fish meal and soybean meal in the feed industry. However, the presence of gossypol residues in both can cause severe intestinal inflammation. At the same time, there are reports showing that gossypol can induce cellular oxidative stress and cell apoptosis, that is, increase the content of reactive oxygen species (ROS) and malondialdehyde (MDA), and reduce the activity of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Inflammation and oxidative stress are important causes of pain. The inventors speculate that Tongluo Qutong Ointment can relieve chronic bone pain by inhibiting the production of gossypol and reducing inflammation.

[0129] In summary, the inventors believe that Tongluo Qutong Paste relieves pain by inhibiting the metabolic process of the above three substances.

[0130] 3.4 Correlation analysis of differential metabolites

[0131] In order to intuitively reflect the closeness of the correlation between the genes with significant differences and the metabolites with significant differences, the inventors conducted a correlation analysis on the genes and metabolites with significant differences in the model group, the injection control group, and the Tongluo Qutong ointment high-dose group ( Figure 1-2 ).

[0132] Figure 1 and Figure 2 Correlation analysis refers to the analysis of two or more variable elements with correlation, so as to measure the degree of correlation between the two variable factors. The degree of correlation between the two variables is represented by the correlation coefficient r. In the present invention, it is the degree of correlation between differential metabolites and differentially expressed genes. The value of the correlation coefficient r is between -1 and 1, but it can be any value within this range. When the correlation is positive, the r value is between 0 and 1; when the correlation is negative, the r value is between -1 and 0. The closer the absolute value of r is to 1, the stronger the correlation between the two variables; the closer the absolute value of r is to 0, the weaker the correlation between the two variables. The horizontal and vertical axes represent the differential metabolites and differential genes compared in this group, and the color blocks in different positions represent the size of the correlation coefficient between the metabolites and genes in the corresponding positions. Red indicates positive correlation, and blue indicates negative correlation. The darker the color, the stronger the correlation. At the same time, significant correlations are marked with asterisks (p<0.05). From Figure 1 and Figure 2As can be seen in the figure, the correlation coefficients r values of the 14 selected metabolites, especially histamine, trimethylamine oxide, gossypol, and piperazine, with most of the differentially expressed genes in the list are close to 1, showing an orange color. This result demonstrates the direct correlation between all the selected differential metabolites and differentially expressed genes.

[0133] 3.5 KEGG enrichment analysis of differential metabolites

[0134] The inventors further performed KEGG pathway enrichment analysis on these 14 differential metabolites.

[0135] The results showed that the pathway enrichment of differential metabolites included the following 9 pathways: metabolic pathways, histidine metabolism, neuroactive ligand-receptor interaction, Fc-εRI signaling pathway, gastric acid secretion, protein digestion and absorption, synaptic vesicle circulation, and inflammatory mediator regulation of TRP channels.

[0136] 3.6 ROC analysis of differential metabolites

[0137] The inventors further drew ROC curves for the 14 clearly identified differential metabolites, namely the ROC curves of the differential metabolites of the model group vs the injection control group and the ROC curves of the differential metabolites of the model group vs the high-dose Tongluo Qutong ointment group, and calculated their area under the curve (AUC).

[0138] The results showed that the average area value under the ROC curve of the 14 metabolites in the model group vs. the injection control group was 0.9024, and the average area value under the ROC curve of the model group vs. the high-dose group of Tongluo Qutong Cream was 0.8785, indicating that the use of the 14 metabolites not only has good accuracy in screening bone pain, but also has good accuracy in detecting the therapeutic effect of Tongluo Qutong Cream.

[0139] Note: The area under the ROC curve ranges from 1.0 to 0.5. When AUC is > 0.5, the closer the AUC is to 1, the better the accuracy. An AUC between 0.5 and 0.7 indicates low accuracy, an AUC between 0.7 and 0.9 indicates moderate accuracy, and an AUC above 0.9 indicates high accuracy. An AUC of 0.5 indicates the method is completely ineffective and worthless.

[0140] 4. Joint analysis of KEGG pathways of transcriptome differentially expressed genes and metabolome differentially expressed metabolites

[0141] To further elucidate the molecular mechanism of Tongluo Pain Relief Paste in treating chronic bone pain, the inventors conducted a joint analysis of transcriptomics pathways enriched with differentially expressed genes and pathways involved in differentially expressed metabolites. They found that differentially expressed genes and metabolites were simultaneously enriched in two pathways: Metabolic pathways and Neuroactive ligand-receptor interaction. This suggests that Tongluo Pain Relief Paste achieves its analgesic effect by simultaneously regulating metabolic-related processes and neuroactive ligand-receptor interactions, reducing the secretion of pain-related metabolites (such as histamine, trimethylamine oxide, and gossypol), and inhibiting the expression of pain-related genes.

[0142] In summary, Tongluo Pain-Relieving Ointment can relieve chronic bone pain by inhibiting the expression of Trpv1, Acod1 and Ebi3 in the joints of rabbits with chronic bone pain, reducing the metabolic levels of histamine, trimethylamine oxide and gossypol, and further reducing the release of inflammatory factors IL-6, IL-1β and TNF-α.

[0143] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for preparing an animal model, comprising: Applying the Tongluo Qutong ointment to the painful area of the individual with pain will produce an animal model having at least one of the following characteristics: D1) Histamine levels decreased; D2) the content of trimethylamine oxide decreased; D3) Gossypol content decreased; D4) 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid levels decreased; D5) 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone content decreased; D6) 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine content decreased; D7) 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine content decreased; D8) 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide content decreased; D9) Benzoic acid,2,4-dimethoxy-6-methyl-,4-carboxy-3-methoxy-5-methylphenyl ester content decreased; D10) Betazole content decreased; D11) Kipukasin A content decreased; D12) Piperazine content decreased; D13) Thiazovivin levels decreased; D14) Warfarin content decreased.

2. A method for preparing an animal model, comprising: By injecting iodoacetic acid into an animal, an animal model having at least one of the following characteristics is obtained: D1) Increased histamine levels; D2) increased trimethylamine oxide levels; D3) increased gossypol content; D4) increased levels of 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid; D5) increased levels of 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone; D6) 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine content increased; D7) increased levels of 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine; D8) increased levels of 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide; D9) Benzoic acid,2,4-dimethoxy-6-methyl-,4-carboxy-3-methoxy-5-methylphenyl ester content increased; D10) Betazole levels increased; D11) Kipukasin A content increased; D12) Piperazine levels increased; D13) Thiazovivin levels increased; D14) Warfarin levels increased.

3. The method according to claim 2, wherein: The injecting of iodoacetic acid into the animal comprises injecting iodoacetic acid into the knee joint cavity of the animal.

4. The method according to claim 2 or 3, characterized in that: The injection amount of iodoacetic acid is 0.012 mg of iodoacetic acid per gram of body weight.

5. The method according to any one of claims 1 to 4, characterized in that: The content of the histamine, trimethylamine oxide, gossypol, 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid, 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone, 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine, 4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine, 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide, Benzoic acid, 2,4-dimethoxy-6-methyl-, 4-carboxy-3-methoxy-5-methylphenyl ester, Betazole, Kipukasin A, Piperazine, Thiazovivin or Warfarin is the content thereof in joint tissue.

6. The method according to any one of claims 1 to 5, characterized in that: The animal model is a rabbit model.

7. At least one of the following uses of a substance for detecting the content of a compound: X1. Use in the preparation of a product for detecting or assisting in detecting the therapeutic effect of Tongluo Qutong Cream on pain, or in detecting or assisting in detecting the therapeutic effect of Tongluo Qutong Cream on pain; X2. Use in the preparation of a product for screening or assisting in screening individuals experiencing pain after treatment with Tongluo Qutong Cream, or use in screening or assisting in screening individuals experiencing pain after treatment with Tongluo Qutong Cream; X3. Use in the preparation of products for screening or assisting in the screening of individuals with pain, or in the screening or assisting in the screening of individuals with pain; The compounds are histamine, trimethylamine oxide, gossypol, 1-(4-Bromophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid, 1,4-Dihydroxy-2-(4-morpholinyl)anthra-9,10-quinone, 2-Linoleoyl-1-palmitoyl-sn-glycero-3-phosphoethanolamine, 4-( 4-Chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine, 4-Amino-N-(2,4-difluorophenyl)benzenesulfonamide, Benzoic acid,2,4-dimethoxy-6-methyl-,4-carboxy-3-methoxy-5-methylphenyl ester, Betazole, Kipukasin A, Piperazine, Thiazovivin or / and Warfarin.

8. The use according to claim 7, characterized in that: The pain is bone pain.

9. The use according to claim 7 or 8, characterized in that: The compound content refers to the content of the compound in joint tissue.

10. The use according to any one of claims 7 to 9, characterized in that: The substance for detecting the compound content also contains a data conversion device, which is used to convert the numerical value of the compound content into the therapeutic effect value of the Tongluo Qutong Paste on pain or the pain value of the individual with pain.

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