Plaster for treating granulomatous lobular mastitis and preparation method thereof

Abstract

The invention discloses a plaster for treating granulomatous lobular mastitis and a preparation method thereof, and relates to the technical field of traditional Chinese medicine, the key points of the technical scheme are as follows: adding water to decoct rheum officinale, golden cypress, scutellaria baicalensis, sanguisorba officinalis, cape jasmine, turmeric and lithospermum erythrorhizon twice, adding water which is 9-11 times of the amount of the medicinal materials for the first time, decocting for 1-3 hours, adding water which is 6-9 times of the amount of the medicinal materials for the second time, decocting for 1-2 hours, and filtering to obtain filtrate; and combining the two water decoctions, filtering, concentrating to obtain ointment, coating, shearing according to specifications, and standing for curing and forming. According to the application disclosed by the invention, the granulometric lobular mastitis can be treated, the index score of mammary inflammation and the infiltration degree of inflammatory cells can be reduced, and the effect of treating the granulometric lobular mastitis is achieved by reducing the expression of IL6, CD86, TYROBP and ITGAX and regulating and controlling related pathways.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and more particularly to a plaster for treating granulomatous lobular mastitis and a preparation method thereof. Background Art

[0002] Granulomatous lobular mastitis (GLM), also known as idiopathic granulomatous mastitis, is a rare inflammatory breast disease of unknown etiology, characterized by non-caseous granulomatous inflammation of the lobules, which may be accompanied by infiltration of lymphocytes, neutrophils, plasma cells, monocytes and eosinophils. It was first described by Kessler E and Wolloch Y in 1972. GLM is not common in clinical practice, especially in European and American countries, where the incidence is low, but has shown an increasing trend year by year in recent years. This disease is more common in women of childbearing age, and is more common in multiparous women with a history of breastfeeding. Some also occur in lactating women, pregnant women or women without a history of childbearing, and a few men can also develop the disease. The clinical manifestations of GLM vary and are diverse. At the beginning of the disease, there are often lumps in the breast, with or without obvious pain. The texture of the lumps is tough and the boundary with the surrounding tissues is unclear. In some cases, the disease progresses rapidly and the lumps may affect the whole breast. In the later stage, suppuration occurs in some parts. In severe cases, multiple sinus tracts or fistulas appear, and pus is dripping, which is difficult to heal. Some patients also have systemic symptoms such as erythema nodosum, fever, and cough in the limbs. Traditional Chinese medicine has not yet made clear records of this disease. According to the characteristics of the disease and clinical manifestations, it can be classified as "breast abscess and milk leakage".

[0003] At present, the western medicine treatment methods of GLM are diverse and controversial, and can be roughly divided into surgical treatment and non-surgical treatment. Traditional Chinese medicine advocates the overall concept, pays attention to the combination of internal and external treatment, and the internal treatment method adopts the external treatment method with unique characteristics of traditional Chinese medicine in due course on the basis of staged treatment, and has achieved a relatively good therapeutic effect. Although this disease is a benign disease, its local invasiveness will cause long-term pain and mental distress to patients. In view of this, the inventor proposes a plaster for treating granulomatous lobular mastitis and a preparation method thereof. Summary of the invention

[0004] The purpose of the invention is to provide a plaster for treating granulomatous lobular mastitis and a preparation method thereof, so as to achieve the treatment of granulomatous lobular mastitis.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a plaster for treating granulomatous lobular mastitis. The raw materials of the plaster for treating granulomatous lobular mastitis are composed of the following ingredients in parts by weight: 13-16 parts of rhubarb, 8-12 parts of phellodendron, 8-12 parts of scutellaria, 13-16 parts of sanguisorba officinalis, 8-12 parts of gardenia, 18-22 parts of turmeric and 13-16 parts of lithospermum officinale.

[0007] In combination with the first aspect, the present invention is further configured as follows: 15 parts of rhubarb, 10 parts of phellodendron, 10 parts of scutellaria, 15 parts of sanguisorba officinalis, 10 parts of gardenia, 20 parts of turmeric, and 15 parts of lithospermum officinale.

[0008] Rhubarb: bitter, cold. Enters the spleen, stomach, large intestine, liver, and pericardium meridians. It has the function of purging and attacking stagnation, clearing away heat and purging fire, cooling blood and detoxifying, removing blood stasis and promoting menstruation, and removing dampness and jaundice. It is used for constipation due to excess heat accumulation, vomiting and bleeding due to blood heat, red eyes and swollen throat, carbuncle, abdominal pain due to intestinal carbuncle, amenorrhea due to blood stasis, postpartum blood stasis, traumatic injury, damp-heat dysentery, jaundice and red urine, stranguria, edema; external treatment for burns and scalds.

[0009] Phellodendron chinense: bitter, cold. Enters the kidney and bladder meridians. Clears away heat and dampness, purges fire and eliminates steam, detoxifies and treats sores. Used for damp-heat diarrhea, jaundice, red urine, vaginal itching, painful stranguria due to heat, beriberi, paralysis, bone steaming, fever due to fatigue, night sweats, spermatorrhea, sores, swelling and poison, eczema and wet sores. Salted Phellodendron chinense nourishes yin and reduces fire. Used for yin deficiency and hyperactivity of fire, night sweats and bone steaming.

[0010] Scutellaria baicalensis: bitter, cold. Enters the lung, gallbladder, spleen, large intestine, and small intestine meridians. Clears away heat and dampness, purges fire and detoxifies, stops bleeding, and stabilizes the fetus. Used for damp-heat, summer-heat, chest tightness and nausea, damp-heat fullness, diarrhea, jaundice, lung heat cough, high fever and thirst, blood heat vomiting and bleeding, carbuncle, sore, and fetal movement disorder.

[0011] Radix Sanguisorbae: bitter, sour, astringent, slightly cold. Enters the liver and large intestine meridians. Cools blood and stops bleeding, detoxifies and heals sores. Used for blood in stool, hemorrhoids, bloody diarrhea, metrorrhagia, burns caused by water or fire, carbuncle, sores and toxicity.

[0012] Gardenia: bitter, cold. Enters the heart, lung, and triple burner meridians. It can relieve heat and relieve restlessness, clear away heat and dampness, cool blood and detoxify; externally used to reduce swelling and relieve pain. It is used for restlessness due to febrile diseases, jaundice due to dampness and heat, astringent pain due to stranguria, vomiting and bleeding due to blood heat, red and swollen eyes, and fire poison sores; external treatment of sprains and contusions.

[0013] Turmeric: pungent, bitter, warm. Enters the spleen and liver meridians. Breaks blood and promotes qi circulation, dredges menstruation and relieves pain. Used for chest and flank pain, chest obstruction and heart pain, dysmenorrhea, amenorrhea, masses, rheumatic shoulder and arm pain, and swelling and pain caused by falls.

[0014] Lithospermum officinale: sweet, salty, cold. Entries to the heart and liver meridians. Heat cools blood, activates blood circulation and detoxifies, clears rashes and eliminates spots. Used for excessive heat and toxicity in the blood, purple and black spots, measles that do not clear up, sores, eczema, and burns caused by water or fire.

[0015] In the formula, rhubarb, phellodendron and scutellaria are the main ingredients, which together play the role of clearing away heat, purging fire, cooling blood and detoxifying; when heat and toxicity are accumulated, the body fluid is refined into phlegm, and the phlegm heat blocks the Qi mechanism and causes Qi and blood to stagnate, "pain occurs when Qi and blood are blocked", and turmeric can promote blood circulation, unblock menstruation and relieve pain, and is the assistant ingredient; Sanguisorba officinalis and Gardenia jasminoides are bitter and cold in nature, and help the main ingredients enhance the power of cooling blood, purging fire and detoxifying; Lithospermum officinale is sweet, salty and cold in nature, and can not only clear away heat, cool blood and detoxify, but can also be combined with turmeric to enhance the power of promoting blood circulation and reducing swelling; the whole formula can both clear away heat, purge fire and detoxify, and promote blood circulation and reduce swelling, and ultimately dissipate the lumps of Yang syndrome.

[0016] The second aspect of the present invention also provides a preparation method of a plaster for treating granulomatous lobular mastitis, comprising the steps of adding water to all the formulated drugs and decocting them twice, adding 9-11 times the amount of water of the medicinal materials for the first time and decocting them for 1-3 hours, adding 6-9 times the amount of water of the medicinal materials for the second time and decocting them for 1-2 hours, combining the two decoctions, filtering and concentrating them to obtain an ointment, applying the ointment, cutting the ointment according to the specifications and placing the ointment to solidify it into a shape.

[0017] In combination with the second aspect, the present invention is further configured as follows: the first decoction time is 2 hours, and the second decoction time is 1.5 hours.

[0018] In combination with the second aspect, the present invention is further configured as follows: the concentration is concentrated at 60° C. to a relative density of 1.15 to 1.18.

[0019] The third aspect of the present invention also provides the use of any one of the above-prepared plasters in the preparation of a drug for treating granulomatous lobular mastitis.

[0020] In combination with the third aspect, the present invention is further configured as follows: the drug is a drug that reduces the expression of IL6, CD86, TYROBP, ITGAX and regulates related pathways.

[0021] In summary, the present invention has the following beneficial effects:

[0022] 1. Through gene sequencing and bioinformatics analysis, and further verification by animal experiments, PTPRC, IL6, IL1B, IL10, CD86, FCGR3A, TYROBP, ITGAX, CXCL8, and SPI1 are potential genes for the onset of granulomatous lobular mastitis. Except for IL10, which is a down-regulated gene, the remaining genes are all up-regulated genes;

[0023] 2. Animal experiments show that Ruyi ointment is effective in treating granulomatous lobular mastitis and can reduce breast inflammation index scores and the degree of inflammatory cell infiltration;

[0024] 3. Ruyi ointment plays a role in treating granulomatous lobular mastitis by reducing the expression of IL6, CD86, TYROBP, and ITGAX and regulating related pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1GO and KEGG pathway analysis of differentially expressed genes in Experimental Example 1 of the present invention (Figure A is a heat map of differentially expressed genes, Control is a normal group, GLM is a granulomatous lobular mastitis group, red represents up-regulated genes, and blue represents down-regulated genes; Figure B is a volcano map of differentially expressed genes, red represents differentially up-regulated genes, and blue represents differentially down-regulated genes; Figure C is a GO analysis diagram of differentially expressed genes; Figure D is a KEGG pathway analysis diagram of differentially expressed genes);

[0026] Figure 2 It is the PPI network diagram of the top ten Hub genes in Experimental Example 1 of the present invention;

[0027] Figure 3 The effect of Ruyi ointment on the macroscopic appearance of rat mammary glands in Experimental Example 2 of the present invention;

[0028] Figure 4 The effect of Ruyi ointment on the pathology of rat mammary gland tissue in Experimental Example 2 of the present invention (HE, ×400);

[0029] Figure 5 This is the effect of Ruyi ointment in Experimental Example 2 of the present invention on the expression level of Hub gene protein in rat mammary tissue. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Embodiment 1:

[0032] Add 15g of rhubarb, 10g of phellodendron, 10g of scutellaria, 15g of sanguisorba officinalis, 10g of gardenia, 20g of turmeric and 15g of lithospermum officinale, and boil them twice with water. For the first time, add 10 times the amount of water of the medicinal materials and boil them for 2 hours. For the second time, add 8 times the amount of water and boil them for 1.5 hours. Combine the two decoctions, filter and concentrate them to a relative density of 1.15-1.18 (60°C), apply them after they become a paste, cut them according to specifications and let them stand to solidify and shape.

[0033] Experimental Example 1 Gene sequencing and Hub gene screening

[0034] 1.1 General Information

[0035] Ten patients who underwent breast biopsy in the Department of Breast Surgery of the First Affiliated Hospital of Hunan University of Chinese Medicine and were diagnosed with granulomatous lobular mastitis and breast fibroadenoma by postoperative pathology were collected. Granulomatous lobular mastitis resection specimens and normal breast tissues were collected during the operation. Written informed consent was obtained from all volunteers for all tissue specimens.

[0036] 1.2 Pathological sampling

[0037] Fresh surgical specimens were collected during the operation, and the collected specimens were quickly frozen in liquid nitrogen and stored in a -80° refrigerator. All the collected specimens were numbered for subsequent testing.

[0038] 1.3 Genetic testing

[0039] 1.3.1 RNA sample testing

[0040] First, RNA was extracted from the sample using a standard extraction method, and then the RNA sample was subjected to strict quality control, mainly including the following three aspects: (1) Concentration quality control: The concentration and total amount were tested using the Qubit4.0 instrument. (2) Fragment integrity quality control: The fragment integrity of the RNA sample was evaluated using the Agilent2100 analyzer. (3) Purity quality control: The sample purity was controlled using the NanoDrop spectrophotometer.

[0041] 1.3.2 mRNA acquisition, library construction and quality control

[0042] Since most eukaryotic mRNAs have polyA tails, Oligo(dT) magnetic beads can be used to enrich these mRNAs with polyA tails. The metal ion solution treatment method is used to select the appropriate shearing temperature and time for mRNA shearing according to the required fragment size. The fragmented mRNA is used as a template, and random oligonucleotides are used as primers in the M-MuLV reverse transcriptase system to synthesize the first cDNA chain. After the synthesis of the first chain is completed, the second chain is synthesized and the end repair and A tail are added. T4 DNA ligase is used to catalyze the connection of the double-stranded DNA sticky ends with short adapters, and AMPureXP beads are used to purify the products after the adapter connection. Finally, cDNA of about 300-350bp is screened, and the concentration of the fragments is ensured. Then PCR amplification is performed, and the sample index tag is added at the same time. Then, AMPure XP beads are used to purify the PCR product to obtain the library. After the library construction is completed, preliminary quantification is performed first. After diluting the library, the Agilent 2100 DNA1000kit is used to detect the distribution of library fragments. After the quality control is qualified, qRT-PCR is used to accurately quantify the effective concentration of the library to ensure the quality of the library.

[0043] 1.3.4 Sequencing and data quality control

[0044] The prepared DNBs were loaded onto the patterned array and PE150 sequencing was performed using the combined probe-anchored polymerization technique (cPAS). After the sequencing primer anchor molecules and fluorescent probes were polymerized on the DNA nanoballs, the light signals were collected, read and identified by the high-resolution imaging system to obtain the single base sequence information, and then the next cycle was entered to obtain the sequence information of the next base. The raw image data file obtained by sequencing with the high-throughput sequencer was converted into the original sequencing sequence after base recognition and analysis, which was called raw data. In order to ensure the quality of information analysis, the software fastp1 was used to remove reads with a length of less than 35bp after the excision of the adapter sequence and low-quality bases at both ends, reads with a ratio of N (N means that the base information cannot be determined) greater than 10% of the total number of bases, and reads with low-quality bases (quality value Q<=5) in single-end reads accounting for more than 40% of the entire read length, and then high-quality clean data was obtained, and the clean data was statistically analyzed for the number of bases and the number of sequences.

[0045] 1.4 Analysis of differentially expressed genes

[0046] After completing the quantitative analysis of genes, the expression levels of all samples were merged into an expression matrix, and the differential expression significance analysis at the gene or transcript level was performed to find functionally differential genes or transcripts related to the sample groups. The DESeq2 software was used for the difference significance analysis, and the corrected Pvalue ≤ 0.001 and |log2FC| ≥ 2.5 were used as the difference significance criteria.

[0047] 1.5 GO and KEGG pathway analysis of differentially expressed genes

[0048] First, all differentially expressed genes were mapped to the relevant entries in the GO database (http: / / www.geneontology.org / ), the number of differentially expressed genes in each entry was counted, and then statistical tests were performed. When the P value ≤ 0.05, it was considered as the standard of significant enrichment, and the GO entries that were significantly enriched in the differentially expressed genes compared with the whole genome background were found. KEGG is the main public database for pathways. The pathway significant enrichment analysis is based on the KEGG pathway, and the hypergeometric test is applied. The P value is less than or equal to 0.05 as the threshold for significant enrichment, and the pathways that are significantly enriched in the differentially expressed genes compared with the whole genome background are found.

[0049] 1.6 PPI network construction and Hub gene identification

[0050] In order to evaluate potential protein-protein interaction relationships, the previously identified differentially expressed genes were mapped to a database for retrieving gene interactions (http: / / string-db.org / ) to construct a protein-protein interaction network (PPI). Subsequently, the PPI network was visualized and analyzed using Cytoscape software, and the top ten genes in the PPI network were selected as key genes (Hub genes) using the Degree algorithm. Hub genes are genes that play a vital role in biological processes. In related signaling pathways, the regulation of other genes is often affected by Hub genes. Hub genes are considered important targets and research hotspots in studies at the gene level.

[0051] Experimental Example 2

[0052] Experimental Materials

[0053] Eighteen female SD rats (250–300 g) were ordered from the Dongtang Experimental Animal Center of Hunan University of Chinese Medicine (Hunan Slake Jingda Experimental Animal Co., Ltd.), license number: SCXK (Xiang) 2019-0004.

[0054] Drugs and reagents

[0055] The plaster prepared in Example 1

[0056] Table 1-1 Drug and reagent information table

[0057]

[0058]

[0059] Homogenate preparation

[0060] The frozen specimens of human granulomatous lobular mastitis tissue were taken out from the -80℃ refrigerator and placed in a 37℃ constant temperature water bath for freeze-thaw. After freeze-thaw, the breast tissue in the cryopreserved tube was taken out and weighed with a precision electronic balance. The tissue was chopped with scissors until there were no obvious large particles, mixed with 0.9% NaCl solution at a ratio of 1:3 and placed in a 15ml centrifuge tube. The sample homogenization system was used to perform cell crushing for about 5 minutes to release the protein in the cytoplasm into the solution. An appropriate amount of the homogenate after cell crushing was placed in a sterile centrifuge tube, and Freund's complete adjuvant was added to the supernatant at a volume ratio of 1:1. The mixture was repeatedly aspirated and mixed with a bus tube to make an oil-in-emulsion suspension, which was stored in a 4℃ refrigerator for later use.

[0061] Experimental methods

[0062] 2.1 Grouping and modeling

[0063] First, 18 rats for modeling were numbered 1 to 18 in sequence, and then randomly divided into three groups using a random number table method, namely a blank group, a model group, and an experimental group (Ruyi cream group), with 6 rats in each group, and adapted to feeding for 3 days. The experimental group and the model group were anesthetized by intraperitoneal injection of 0.2 mL / 100 g of a 2% pentobarbital solution. After successful anesthesia, the third and fourth pairs of breasts and surrounding hair were removed with a depilatory cream, the nipples and nearby skin were disinfected with iodine, and 0.2 ml of the suspension prepared in advance was extracted with a 1 ml syringe and injected into the third and fourth pairs of mammary glands of the rats, respectively, to complete the artificial implantation and embedding process. After the needle was removed, simple pressing was performed to stop bleeding; the rats in the normal group were not treated.

[0064] Verification of the granulomatous lobular mastitis model: Through preliminary experimental verification, before the experiment began, 5 SD rats were used to establish the model using the above method, and the general conditions were observed and recorded. After obvious local lumps appeared, the model rats were killed and samples were obtained for pathological observations such as HE staining. Granuloma-like lesions appeared, and the general condition was manifested as local redness and swelling (positive syndrome model), indicating that the model was successfully established.

[0065] 2.2 Administration

[0066] After successful modeling, the experimental group was given 4cm×3cm Ruyi ointment on the third and fourth pairs of breasts, and the control group (normal group and model group) was not treated. After administration, the mice were fixed with an external bandage. The frequency of administration was once a day for 14 consecutive days.

[0067] 2.3 General Observations

[0068] After modeling and drug administration, rats in each group were maintained in a routine manner. The performance of rats in each group was observed every day, and the changes of animals in each group were recorded, including the rat status, whether the breast was red and swollen, whether abscesses were formed, skin color, etc.; and scores were given according to the breast inflammation index score table based on the rat performance.

[0069] Table 1-2 Breast Inflammation Index Scoring Table

[0070]

[0071]

[0072] 2.4 Collection of materials

[0073] On the 14th day after administration, all rats were killed, and the 3rd and 4th pairs of mammary gland tissues were removed. One part was rinsed with saline and fixed in 4% paraformaldehyde solution, and the other part was stored in a -80°C refrigerator for later RT-qPCR and Western blot experiments.

[0074] 2.2.2.5 Observation indicators and indicator detection

[0075] (1) The local and overall manifestations of the mammary glands of rats in each group were observed and scored according to the mammary inflammation index scoring table set up above. The final mammary inflammation index was the sum of the scores of each item.

[0076] (2) The breast tissue was routinely paraffin-embedded and sliced, with a slice thickness of 4 μm. The intact breast tissue on the slice was selected for dewaxing, HE staining, and finally dehydration and sealing. The morphological characteristics of the breast tissue and the infiltration of inflammatory cells were observed under an optical microscope.

[0077] (3) Western blot kit was used to detect the expression level of the target gene protein in the above gene sequencing in each tissue sample: appropriate amount of rat mammary tissue was cut, RIPA lysis buffer was added for lysis and centrifugation to obtain protein supernatant, protein denaturation was performed while gel preparation was performed, electrophoresis was started after gel filling and sample loading, filter paper and NC membrane were first placed in transfer buffer for transfer, and then the membrane was soaked in 5% skim milk powder prepared with PBST and allowed to stand at room temperature for 90 minutes; then the primary antibody was diluted with PBST according to a certain ratio and incubated with the membrane at 4°C overnight, and allowed to stand at room temperature for 30 minutes the next day, and then the HRP-labeled secondary antibody was diluted with PBST, and the diluted secondary antibody was incubated with the membrane at room temperature for 90 minutes. Finally, the membrane was incubated with ECL chemiluminescent solution for 1 minute, and then the membrane was wrapped with plastic film and placed in a gel imaging system for imaging.

[0078] (4) Real-time PCR detection of potential target gene expression: The total RNA was extracted from rat mammary tissue using the Trizol method, and the concentration of RNA was determined using an ultraviolet spectrophotometer, and its concentration and purity were calculated. The total tissue mRNA was used as a template for reverse transcription to generate cDNA, and SYBR fluorescent dye and upstream and downstream primers were added to perform real-time quantitative PCR experiments (3 reaction wells for each sample and each indicator, with a total volume of 30uL, 10uL per well), and finally a quantitative PCR amplification procedure was performed. The target gene sequence was retrieved from the NCBI database, and primers were designed using primer5 software, and then synthesized by Beijing Qingke. The specific primer information is shown in the following table:

[0079] Table 1-4 Primer sequences and primer lengths

[0080]

[0081] Note: The primer sequence of CXCL8 was not found in NCBI, so real-time PCR was not performed on it.

[0082] 2.3 Statistical methods

[0083] SPSS 25.0 software was used for statistical analysis, and GraphPad Prism 9.0 was used to draw statistical charts. When normality and variance homogeneity were met, one-way ANOVA was used to compare the differences between the groups. When normality and / or variance homogeneity were not met, Kruskal-Wallis H test was used to compare the differences between the groups. P < 0.05 was set as statistically significant.

[0084] 3 Experimental results

[0085] 3.1 GO and KEGG pathway analysis of differentially expressed genes

[0086] Using Pvalue≤0.001 and |log2FC|≥2.5 as the difference significance criteria, the sequencing data were analyzed by DESeq2 software, and a total of 1210 differentially expressed genes (DEGs) were screened, including 1095 up-regulated genes and 115 down-regulated genes. The DEGs of the dataset were visualized ( Figure 1 A, B). GO and KEGG pathway analysis were performed on the identified differentially expressed genes ( Figure 1C, D), GO analysis showed that biological process (BP) was mainly enriched in leukocyte mediated immunity, positive regulation of cell activation, and positive regulation of leukocyte activation; cellular component (CC) was mainly enriched in the external side of plasma membrane, plasma membrane receptor complex, and immunoglobulin complex; molecular function (MF) was mainly enriched in antigen binding, immune receptor activity, and cytokine activity. KEGG pathway analysis showed that DEGs were enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway, and viral protein interaction with cytokine and cytokine receptor.

[0087] Construction of PPI network of differentially expressed genes and identification of hub genes

[0088] The 1210 differentially expressed genes (DEGs) screened were imported into the string website to obtain the PPI network, and the top ten genes (Hub genes) were selected by the Degree algorithm, which were PTPRC, IL6, IL1B, IL10, CD86, FCGR3A, TYROBP, ITGAX, CXCL8, and SPI1 ( Figure 2 ).

[0089] Ruyi ointment reduces breast inflammation index in GLM model rats

[0090] Depend on Figure 3As shown in Table 2-1, compared with the normal group, the breast inflammation index of rats in the model group increased (P < 0.05); compared with the model group, the breast inflammation index of rats in the experimental group after treatment with Ruyi ointment decreased (P < 0.05).

[0091] Table 2-1 Effect of Ruyi ointment on rat breast inflammation index

[0092]

[0093] Note: 1) Compared with the blank group, 2) Compared with the model group.

[0094] Ruyi ointment reduces the infiltration of inflammatory cells in mammary gland tissue of GLM model rats

[0095] In normal mammary tissue of rats, the mammary lobules and ducts were clearly structured, and the cells were arranged relatively tightly. Compared with the blank group, in the mammary tissue of rats in the model group, lesions involving the terminal duct lobules were observed, and the outlines of most lobule structures were blurred and difficult to distinguish. Lymphocytes, plasma cells and macrophages were infiltrated, and epithelial-like cells aggregated to form granulomas in most lobules in the lesions. Compared with the model group, the experimental group could reduce the infiltration of inflammatory cells such as lymphocytes and plasma cells, and reduce the formation of inflammatory granulomas ( Figure 4 ).

[0096] Ruyi ointment affects the mRNA expression of some Hub genes

[0097] As can be seen from Table 2-2, compared with the blank group, the relative expression levels of PTPRC, IL6, IL1B, CD86, FCGR3A, TYROBP, ITGAX, and SPI1 mRNA in the mammary gland tissue of rats in the model group were significantly increased (P < 0.001), while the relative expression level of IL10 mRNA was significantly decreased (P < 0.001), which had significant statistical differences; compared with the model group, the experimental group could significantly down-regulate the expression of PTPRC, IL6, IL1B, CD86, FCGR3A, TYROBP, ITGAX, and SPI1 mRNA (P < 0.01), and up-regulate the expression of IL10 mRNA (P < 0.01).

[0098] Table 2-2 Effect of Ruyi ointment on relative expression of Hub gene mRNA in rat mammary gland tissue (2 -ΔΔCt , X±SD, n=3)

[0099]

[0100] Note: Compared with the blank group, 1) P<0.001; compared with the model group, 2) P<0.01.

[0101] 3.6 Ruyi ointment affects the protein expression of some Hub genes

[0102] Compared with the blank group, the expression of PTPRC, IL6, IL1B, CD86, FCGR3A, TYROBP, ITGAX, CXCL8, and SPI1 proteins in the mammary gland tissue of the rats in the model group was significantly increased (P < 0.01), while the protein expression of IL10 was decreased compared with the blank group (P < 0.05); compared with the model group, the protein expression of IL6, CD86, TYROBP, and ITGAX in the experimental group was decreased (P < 0.05), and there was no significant difference in the protein expression of the other Hub genes. Figure 5 , Table 2-3)

[0103] Table 2-3 Effect of Ruyi ointment on the expression of Hub gene protein in rat mammary tissue (X±SD, n=3)

[0104]

[0105] Note: Compared with the blank group, 1) P<0.01, 2) P<0.05; compared with the model group, 3) P<0.05

[0106] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A plaster for treating granulomatous lobular mastitis, characterized in that: The raw materials of the plaster for treating granulomatous lobular mastitis are composed of the following ingredients by weight: 13-16 parts of rhubarb, 8-12 parts of phellodendron, 8-12 parts of scutellaria, 13-16 parts of sanguisorba officinalis, 8-12 parts of gardenia, 18-22 parts of turmeric and 13-16 parts of lithospermum officinale.

2. A plaster for treating granulomatous lobular mastitis according to claim 1, characterized in that: 15 parts of Rheum officinale, 10 parts of Cortex Phellodendri, 10 parts of Radix Scutellariae, 15 parts of Radix Sanguisorbae, 10 parts of Fructus Gardeniae, 20 parts of Curcuma Longa and 15 parts of Radix Lithospermi.

3. A method for preparing a plaster for treating granulomatous lobular mastitis according to any one of claims 1 to 2, characterized in that: All the formulated drugs are decocted twice with water. For the first time, 9-11 times the amount of water of the medicinal materials are added and decocted for 1-3 hours. For the second time, 6-9 times the amount of water of the medicinal materials are added and decocted for 1-2 hours. The two decoctions are combined, filtered and concentrated to obtain an ointment, which is then applied and cut according to specifications and placed to solidify into shape.

4. A method for preparing a plaster for treating granulomatous lobular mastitis according to claim 3, characterized in that: The first decoction time is 2 hours, and the second decoction time is 1.5 hours.

5. A method for preparing a Chinese medicine composition for treating granulomatous lobular mastitis according to claim 3, characterized in that: The concentration is carried out at 60° C. to a relative density of 1.15 to 1.

18.

6. Use of the patch according to any one of claims 1 to 2 or the patch prepared by the preparation method according to claims 3 to 5 in preparing a drug for treating granulomatous lobular mastitis.

7. Use of a plaster according to claim 6 in preparing a drug for treating granulomatous lobular mastitis, characterized in that: The drug is a drug that reduces the expression of IL6, CD86, TYROBP, and ITGAX and regulates related pathways.

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