A method for verifying a traditional Chinese medicine compound for treating mycoplasma synoviae infection in chickens

Abstract

The application discloses a traditional Chinese medicine compound regulation verification method for treating chicken mycoplasma synoviae infection, and relates to the technical field of traditional Chinese medicine treatment. The method is used for studying the treatment and regulation effects of a traditional Chinese medicine compound on chicken mycoplasma synoviae disease, and comprises the following steps: determining the treatment effect of the traditional Chinese medicine compound dry powder on MS infected cells, detecting key inflammatory factors and key proteins, and determining the recovery regulation effect of the traditional Chinese medicine compound dry powder on the autophagy pathway. The traditional Chinese medicine compound dry powder is prepared from Huangqi, Huangbai, Huangqin, Danggui, Juhua, Huaihua, Sangye, Yinxingye and Pugongying. The traditional Chinese medicine compound dry powder has a multi-target and multi-pathway action mechanism, and can synergistically regulate the immune homeostasis in the MS infection process. By inhibiting the inflammatory reaction, recovering the expression of anti-inflammatory factors, and recovering the autophagy flow to promote the cleaning of pathogens and damaged cells in the body, the traditional Chinese medicine compound can achieve the purpose of double regulation and treatment of chicken mycoplasma synoviae inflammation.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine treatment technology, specifically to a method for regulating and verifying a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens. Background Technology

[0002] Mycoplasma synoviae (MS) is a highly infectious and economically important pathogen in poultry farming. This pathogen primarily infects the respiratory system and synovial bursae of chickens, causing mycoplasma synoviae infection, leading to stunted growth, decreased egg production in laying hens, and reduced eggshell quality. Currently, antimicrobial drugs such as tylosin and doxycycline are commonly used to treat MS infection inflammation. However, long-term use of these drugs leads to drug resistance in MS, reducing their effectiveness. Furthermore, antimicrobial drugs are easily incompletely metabolized in poultry, increasing compound residues and compromising food safety. Moreover, the therapeutic mechanisms of drugs against MS infection inflammation are currently difficult to elucidate. This application establishes an MS infection model to verify the multiple targets and pathways of a traditional Chinese medicine (TCM) compound in treating MS infection inflammation, and clarifies the dual regulatory effects of the TCM compound powder on immune inflammation and autophagy pathway regulation, thereby effectively achieving the therapeutic effect of the TCM compound powder on MS infection inflammation. Summary of the Invention

[0003] The purpose of this invention is to provide a method for regulating and verifying a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens, so as to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for verifying the regulatory effects of a traditional Chinese medicine compound on Mycoplasma synoviae infection in chickens, used to study the therapeutic and regulatory effects of the traditional Chinese medicine compound on Mycoplasma synoviae disease in chickens. The traditional Chinese medicine compound is a dry powder obtained by extraction, filtration, concentration, and freeze-drying of Astragalus membranaceus, Phellodendron chinense, Scutellaria baicalensis, Angelica sinensis, Chrysanthemum morifolium, Sophora japonica, Morus alba leaves, Ginkgo biloba leaves, and Taraxacum mongolicum, comprising the following steps: Step 1: Prepare the traditional Chinese medicine compound powder and establish a cell model of Mycoplasma synoviae (MS) infection in chickens; Step 2: Determine the therapeutic effect of the compound Chinese medicine powder on MS-infected cells; Step 3: Detection of key inflammatory factors to clarify the inhibitory and regulatory effects of the traditional Chinese medicine compound powder on the excessive production of key pro-inflammatory factors and the restorative and enhancing regulatory effects on the expression of key anti-inflammatory factors; Step 4: Detection of key proteins to clarify the regulatory role of traditional Chinese medicine compound powder in the restoration of autophagy pathway; Step 5: Functional recovery experiment to verify the regulatory effect of traditional Chinese medicine compound powder on autophagic flux and the correlation between autophagy pathway and anti-inflammatory treatment.

[0005] Preferably, the establishment of the cell model of Mycoplasma synoviae infection in chickens includes: HD11 cells or primary chicken synovial cells were selected as cells to be infected. Cells in the logarithmic growth phase were seeded into multi-well culture plates and cultured at 37°C and 5% CO2 until they adhered to the plate. The culture system was then randomly divided into two groups: the infected system and the control system. The standard strain of Mycoplasma synoviae and an equal volume of cell culture medium were added to the infected system and the control system, respectively. The two systems were then cultured at 37°C and 5% CO2 to obtain the MS infection group and the control group. The establishment status of the MS infection cell model was detected by real-time PCR. The MS-specific nucleic acid and MS-specific DNA copy numbers in the two systems were detected. When the MS-specific nucleic acid detection result in the MS-infected group was positive, the MS-specific nucleic acid detection result in the judgment control group was negative, and the MS-specific DNA copy number in the MS-infected group was higher than that in the judgment control group, it indicated that the MS-infected group had successfully established a cell model of MS infection.

[0006] Preferably, the preparation of the traditional Chinese medicine compound dry powder includes, by weight percentage, the following traditional Chinese medicine raw materials: Astragalus membranaceus 10-20%, Phellodendron chinense 5-15%, Scutellaria baicalensis 8-15%, Angelica sinensis 5-12%, Chrysanthemum morifolium 8-15%, Sophora japonica 6-15%, Morus alba 8-15%, Ginkgo biloba 5-12%, and Taraxacum mongolicum 8-15%; The raw materials of traditional Chinese medicine were crushed and mixed separately. Then, the crude extract of the Chinese medicine was obtained by decoction or alcohol extraction. The crude extract was filtered or rotary evaporated to remove the alcohol solution, and the second extract was obtained. The second extract was concentrated and dried to obtain the compound powder of traditional Chinese medicine. The compound powder of traditional Chinese medicine was stored at -20℃ in the dark.

[0007] Preferably, the evaluation of the therapeutic effect of the traditional Chinese medicine compound powder on MS-infected cells; This includes mixing traditional Chinese medicine compound dry powder with DMSO to prepare a treatment stock solution, and then adding cell culture medium to dilute the treatment stock solution to obtain traditional Chinese medicine compound treatment solutions with concentrations of 25 μg / mL, 50 μg / mL and 100 μg / mL; For the successfully established MS infection cell model obtained in step one, they were randomly divided into a model control group, a traditional Chinese medicine compound treatment liquid group, and a positive drug control group. The judgment control group in step one was used as the blank control group in this step. 25 μg / mL, 50 μg / mL and 100 μg / mL of traditional Chinese medicine compound treatment liquid were added to the traditional Chinese medicine compound treatment liquid group and tylosin was added to the positive drug control group and cultured for 24 h. Cell viability in the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group after culture was detected by CCK-8 or MTT assay. Cell viability in the blank control group was used as the control standard and judgment criteria were set. Cell viability in the traditional Chinese medicine compound treatment liquid group was compared with the judgment criteria to obtain the concentration of non-cytotoxic traditional Chinese medicine compound treatment liquid. Supernatants from the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group were collected after culture. The 16S rRNA gene copy number of MS in the supernatant of each group was detected by real-time quantitative PCR. Combined with the cell viability detection results, the therapeutic effect of traditional Chinese medicine compound powder on MS-infected cells was clarified.

[0008] Preferably, the key inflammatory factors include key pro-inflammatory factors and anti-inflammatory factors; The detection of key inflammatory factors includes collecting cells and their supernatants from the cultured blank control group, model control group, traditional Chinese medicine compound treatment liquid group and positive drug control group, and using enzyme-linked immunosorbent assay (ELISA) to detect the protein secretion levels of key pro-inflammatory and anti-inflammatory factors in the supernatant. Total RNA was extracted from cells and their supernatants in each experimental group. The relative expression levels of key inflammatory factors mRNA in cells were detected by real-time quantitative PCR. The detection results of the blank control group, model control group, traditional Chinese medicine compound treatment liquid group and positive drug control group were compared to analyze the regulatory effect of traditional Chinese medicine compound powder on pro-inflammatory factors induced by MS infection and the correlation between the concentration of traditional Chinese medicine compound treatment liquid and the expression level of key inflammatory factors.

[0009] Preferably, the key protein detection includes collecting total cellular protein from the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group after culture, and using protein immunoblotting to detect the expression levels of key autophagy marker proteins LC3-Ⅰ, LC3-Ⅱ, Beclin-1, ATG5, ATG7, and p62, and analyzing the autophagy flux by comparing the LC3-Ⅱ / LC3-Ⅰ ratio and changes in p62 expression. Based on the detection of autophagy regulatory pathway and inflammation pathway using genomics, protein immunoblotting was used to detect the phosphorylation and expression levels of AKT and mTOR proteins in the autophagy regulatory pathway and inflammation pathway, as well as the expression levels of IκBα and p65 proteins in the inflammation pathway. The regulatory effect of traditional Chinese medicine compound treatment liquid on autophagy pathway was analyzed based on the detection results.

[0010] Preferably, the analysis of the autophagy flux state includes the following: when the LC3-II / LC3-I ratio increases and the p62 protein expression level decreases in the traditional Chinese medicine compound treatment liquid group, it indicates that the traditional Chinese medicine compound treatment liquid inhibits the overactivation of the autophagy regulatory pathway and restores the autophagy flux state.

[0011] Preferably, the detection of key inflammatory factors and key proteins further includes collecting cells and their supernatants from the traditional Chinese medicine compound treatment liquid group, the positive drug control group with added tylosin, the blank control group, and the model control group at 6h, 12h, 24h, and 48h of culture, respectively. The transient changes in the relative expression levels of key inflammatory factor mRNA are detected in step three, and the autophagic flux status and the phosphorylation level of proteins in the inflammatory pathway are detected in step four.

[0012] Preferably, the functional recovery experiment includes taking the successfully established MS infection cell model obtained in step one, randomly dividing it into an infection model control group, a traditional Chinese medicine compound treatment group, an autophagy inhibitor treatment group, and a traditional Chinese medicine compound treatment group combined with an autophagy inhibitor treatment group, and culturing it under the conditions in step two. Then, the cell survival rate and expression level of key inflammatory factors in each experimental group are detected by step two and step three to verify the key molecular mechanism of traditional Chinese medicine compound regulating MS infection inflammatory factors and autophagy flux recovery, and to clarify the association between autophagy pathway and MS infection inflammation.

[0013] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention prepares a traditional Chinese medicine compound powder using Astragalus membranaceus, Phellodendron chinense, Scutellaria baicalensis, Angelica sinensis, Chrysanthemum morifolium, Sophora japonica, Morus alba, Ginkgo biloba, and Taraxacum mongolicum. Utilizing the multi-target and multi-pathway mechanism of this compound powder, it synergistically regulates immune homeostasis during MS infection. By inhibiting inflammatory responses and restoring the expression of anti-inflammatory factors, as well as restoring autophagic flux to promote cellular clearance of pathogens and damaged cells, the invention achieves a dual therapeutic effect of the traditional Chinese medicine compound on Mycoplasma synoviae inflammation in chickens.

[0014] 2. This invention elucidates the regulatory effect of traditional Chinese medicine compound powder on the inflammatory immune homeostasis of Mycoplasma synoviae in chickens, as well as the regulatory mechanism of autophagy pathway and inflammatory immune homeostasis. By correcting the target of action of the traditional Chinese medicine compound powder to chicken origin, the applicability of the predicted target of action of the traditional Chinese medicine compound powder to Mycoplasma synoviae inflammation in chickens is improved, providing an accurate basis for the treatment of Mycoplasma synoviae inflammation in chickens with traditional Chinese medicine compound powder.

[0015] 3. This invention, by dynamically monitoring the autophagic flux status, expression levels of key inflammatory factors, and phosphorylation and expression levels of key proteins in the pathway, facilitates the elucidation of the synergistic effects of key inflammatory factor regulation and autophagy pathway regulation in the treatment of Mycoplasma synoviae inflammation in chickens. This indicates that the traditional Chinese medicine compound powder has a synergistic regulatory effect on inflammatory factors and autophagy pathways in the treatment of Mycoplasma synoviae in chickens. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific implementation methods. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] One embodiment of the present invention is a method for verifying the regulation of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens. This method is used to study the therapeutic and regulatory effects of the traditional Chinese medicine compound on Mycoplasma synoviae infection in chickens. The traditional Chinese medicine compound is a dry powder obtained by extracting, filtering, concentrating, and freeze-drying Astragalus membranaceus, Phellodendron chinense, Scutellaria baicalensis, Angelica sinensis, Chrysanthemum morifolium, Sophora japonica, Morus alba, Ginkgo biloba, and Taraxacum mongolicum. The preparation of the traditional Chinese medicine compound powder includes taking 15% Astragalus membranaceus, 10% Phellodendron chinense, 12% Scutellaria baicalensis, 8% Angelica sinensis, 12% Chrysanthemum morifolium, 10% Sophora japonica, 12% Morus alba leaf, 9% Ginkgo biloba leaf, and 12% Taraxacum mongolicum by weight percentage. The raw materials are then pulverized and mixed. A crude extract is obtained using either a decoction-water extraction method or an alcohol extraction method. The crude extract is then filtered, concentrated, or subjected to rotary evaporation to remove the alcohol solution, yielding a second extract. The decoction-water extraction method involves soaking the pulverized medicinal materials in water for 40 minutes, with the water volume equal to the amount of medicinal materials. The volume ratio was 10:1. The Chinese medicinal materials were decocted for the first time, and the water volume was reduced to 1 / 5 of the original water volume. Then, water with a volume of 7 times that of the Chinese medicinal materials was added to the decoction set, and the second decoction was carried out. After decocting for 1 hour, the decoction was stopped and cooled to room temperature. The Chinese medicinal materials were then filtered and concentrated using a vacuum concentration and decompression equipment at 50℃ and 0.08MPa vacuum to obtain a crude extract of Chinese medicinal materials. The extract was then freeze-dried at -45℃ for 24 hours to obtain a compound Chinese medicine powder. The obtained compound Chinese medicine powder was stored at -20℃. The verification methods for the regulation of traditional Chinese medicine compound prescriptions also include revealing the multi-target mechanism of action of traditional Chinese medicine compound dry powder in treating mycoplasma synovitis in chickens based on network pharmacology and molecular docking technology; This includes establishing a database of chemical components of traditional Chinese medicine (TCM) compound formulas. The process involves mixing TCM compound powder, water, and methanol to obtain a compound powder solution. This solution is then subjected to ultrasonic extraction, centrifugation, and filtration of the supernatant. The TCM compound powder samples are analyzed using LC-MS / MS to obtain the total ion chromatogram, characteristic peak retention time, primary mass spectrometry molecular ion peak, secondary mass spectrometry fragment ion, and relative peak area. The detected molecular ion peak, secondary fragment ion, retention time, and relative peak area are compared with known chemical component information of TCM compound powder raw materials in the TCM systems pharmacology database and analysis platform and the TCM integrated pharmacology research platform to identify effective chemical components for inflammation treatment in the TCM compound powder. A TCM compound chemical component database is established, including compound name, molecular formula, structural formula, molecular weight, retention time, characteristic fragment ion, relative peak area, and known pharmacological activity. This provides a data foundation for subsequent screening of active ingredients in TCM compound formulas and predicts the targets of active ingredients. To avoid false positive predictions that are made solely based on the theoretical components of traditional Chinese medicine; Predicting the target sites for active ingredients in traditional Chinese medicine (TCM) compound powders involves: based on drug-likeness analysis and oral bioavailability models, setting screening criteria as oral bioavailability of no less than 30%, drug-likeness index of no less than 0.2, and stable peak values; preliminary screening of chemical components in the TCM compound chemical component database to obtain compounds with good in vivo absorption and distribution characteristics as potential active ingredients in the TCM compound powder; inputting the standardized molecular formulas and chemical structures of the potential active ingredients into SwissTargetPrediction, PharmMapper, and SEA prediction platforms to obtain initial targets; combining, standardizing, and deduplicating the initial targets obtained from different prediction platforms to obtain an initial target set; converting the initial targets into standard protein names and standard gene names; and using EnsemblBioMart, NCBI Gene, and NCBI... A method using the HomoloGene database to map chicken homologous genes and screen for targets homologous to chicken proteins, with screening criteria including sequence identity of at least 75%, conserved domains, and functional annotations in inflammatory factors and autophagy transformation reactions, yields a set of chicken-derived regulatory targets. By correcting the initial target set for chicken-derived characteristics, the predicted targets of active ingredients in traditional Chinese medicine compound powders are matched with Mycoplasma synoviae infection targets in chickens. This improves the correlation between the predicted targets of traditional Chinese medicine compound powders and Mycoplasma synoviae inflammation, thereby enhancing the therapeutic effect of traditional Chinese medicine compound powders on inflammation. Based on the keyword search of mycoplasma synoviae disease in chickens, avian arthritis, and synovitis, target genes for mycoplasma synoviae disease in chickens were obtained. The gene databases included GeneCards, OMIM, and DisGeNET. The intersection of the target genes and the set of regulatory targets was processed to obtain the potential direct targets of the traditional Chinese medicine compound dry powder for the treatment of mycoplasma synoviae disease in chickens, which were recorded as common targets. Using active ingredients and targets as nodes and interaction relationships as edges, a network graph of "active ingredients of traditional Chinese medicine compound dry powder - common targets of disease" was constructed using Cytoscape software. The active ingredients and key targets with high connectivity in the network graph were identified by network analysis. The common targets were imported into bioinformatics analysis tools for GO and KEGG enrichment analysis to identify the biological processes, cellular components, biological functions, and enrichment pathways of the target genes. The common targets were enriched in the PI3K-Akt pathway, mTOR pathway, NF-κB pathway, Toll-like receptor signaling pathway, and autophagy-related pathway, and the key regulatory pathways of the active ingredients were obtained. The three-dimensional crystal structures of core target proteins in key regulatory pathways were obtained from protein databases and processed using molecular docking technology. The three-dimensional chemical structures of potential active ingredients were obtained from the PubChem database, and the binding free energy between potential active ingredients and core target proteins was calculated to obtain the binding ability between potential active ingredients and key target proteins. This revealed that the traditional Chinese medicine compound powder can achieve multi-target action to treat mycoplasma synoviae disease in chickens and provided a data foundation for subsequent binding of key inflammatory factors and detection of key proteins. A comprehensive model of components, targets, and pathways was established. Based on the model score, the core active ingredients and core targets for inflammation treatment of the traditional Chinese medicine compound powder were identified. The model score was calculated as follows: ; Based on the scoring values, the top-scoring core active ingredients and core targets are selected. The core active ingredients act on the core targets to treat mycoplasma inflammation in chicken synovial bursae. Where H is the measured weight of the active ingredient in the traditional Chinese medicine compound, D is the pharmacokinetic characteristic weight, B is the target prediction weight, W is the network topology weight and J is the binding ability weight, and h, d, b, w, and j are the corresponding weight coefficients, and the sum of h, d, b, w, and j is 1.

[0018] One embodiment of the present invention provides a method for regulating and verifying a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens, comprising the following steps: constructing a cell model of Mycoplasma synoviae (MS) infection in chickens. The in vitro infection model is obtained through in vitro infection, specifically including: selecting HD11 cells as cells to be infected, seeding cells in the logarithmic growth phase into multi-well culture plates and adding complete culture medium, and culturing the cells to be infected to adherence at 37°C and 5% CO2 concentration to obtain a culture system. The culture system is randomly divided into two groups to obtain a to-infect system and a judgment control system. Then, a standard strain of Mycoplasma synoviae and an equal volume of cell culture medium are added to the to-infect system and the judgment control system, respectively, with the multiplicity of infection (MOI) of the standard strain of Mycoplasma synoviae being 10:1. The two systems are cultured at 37°C and 5% CO2 to obtain an MS infection group and a judgment control group. The establishment status of the MS infection cell model is detected by real-time PCR technology. Furthermore, real-time quantitative PCR was used to detect the copy numbers of MS-specific nucleic acid and MS-specific DNA in the cultured anti-infection system and the judgment control system. When the MS-specific nucleic acid detection result in the MS-infected group was positive and the MS-specific nucleic acid detection result in the judgment control group was negative, and the MS-specific DNA copy number in the MS-infected group was higher than that in the judgment control group, it indicated that the MS-infected cell model was successfully established. By establishing the MS-infected cell model, a receptor was provided to verify the regulatory effect of the traditional Chinese medicine compound powder on the treatment of Mycoplasma synoviae inflammation in chickens.

[0019] One embodiment of the present invention is a method for regulating and verifying a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens, including step two: clarifying the therapeutic effect of the traditional Chinese medicine compound powder on MS-infected cells, which also includes evaluating the effect of the traditional Chinese medicine compound powder on the viability of MS-infected cells and its inhibitory effect on pathogens; Traditional Chinese medicine compound powder was mixed with DMSO to prepare a treatment stock solution. Cell culture medium was then added to the treatment stock solution for dilution, resulting in traditional Chinese medicine compound treatment solutions with concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL. By preparing treatment solutions with three concentration gradients of traditional Chinese medicine compound powder, it is convenient to verify the effect of the concentration of traditional Chinese medicine compound powder on cell activity. This avoids the problem of insufficient treatment or damage to poultry cells due to the influence of drug concentration when applying traditional Chinese medicine compound powder to treat mycoplasma synoviae inflammation in live poultry. For the MS infection cell model successfully established in step one, the cells were randomly divided into a model control group, a traditional Chinese medicine compound treatment liquid group, and a positive drug control group. The judgment control group in step one was used as the blank control group in this step. That is, the blank control group in this step did not include the standard strain of Mycoplasma synoviae or other substances with therapeutic effects. 25 μg / mL, 50 μg / mL and 100 μg / mL of traditional Chinese medicine compound treatment liquid were added to the traditional Chinese medicine compound treatment liquid group, and tylosin was added to the positive drug control group. Then, the cells were cultured for 24 h. Cell viability in the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group after culture was detected by CCK-8 or MTT assay. Cell viability in the blank control group was used as the control standard and judgment criteria were set. Cell viability in the traditional Chinese medicine compound treatment liquid group was compared with the judgment criteria to obtain the concentration of non-cytotoxic traditional Chinese medicine compound treatment liquid. Supernatants from the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group were collected after culture. The 16S rRNA gene copy number of MS in the supernatant of each group was detected by real-time quantitative PCR. Combined with the cell viability detection results, the therapeutic effect of traditional Chinese medicine compound powder on MS-infected cells was clarified. When the cell survival rate in the herbal compound dry powder treatment group was higher than 90% of that in the blank control group, it indicated that the corresponding concentration of the herbal compound dry powder treatment solution in the herbal compound dry powder treatment group had no cytotoxicity. When the cell survival rate in the herbal compound dry powder treatment group was 75%–90% of that in the blank control group, it indicated that the concentration of the herbal compound dry powder treatment solution had slight toxicity. Combined with the detection results of MS 16S rRNA gene copy number, when the concentration of the herbal compound dry powder treatment solution had no cytotoxicity, the gene copy number was lower than that in the model control group, and the gene copy number decreased in a concentration-dependent manner with the concentration of the herbal compound dry powder treatment solution, it indicated that the herbal compound dry powder treatment solution had an inhibitory effect on the proliferation of MS pathogens in cells and released into the supernatant.

[0020] One embodiment of the present invention provides a method for verifying the regulation of a traditional Chinese medicine compound in treating Mycoplasma synoviae infection in chickens, including step three: detection of key inflammatory factors to clarify the inhibitory and regulatory effects of the traditional Chinese medicine compound dry powder on the excessive production of key pro-inflammatory factors and the restorative and enhancing regulatory effects on the expression of key anti-inflammatory factors. The key inflammatory factors are the potential active ingredients in the traditional Chinese medicine compound dry powder treatment solution and include key pro-inflammatory factors and key anti-inflammatory factors. The detection of key inflammatory factors included collecting cells and their supernatants from the blank control group, model control group, traditional Chinese medicine compound treatment liquid group and positive drug control group after culture, and using enzyme-linked immunosorbent assay to detect the protein secretion levels of key pro-inflammatory and anti-inflammatory factors in the supernatant. Total RNA was extracted from cells and their supernatants in each experimental group. The relative expression levels of key inflammatory factors mRNA in cells were detected by real-time quantitative PCR. The results of the blank control group, model control group, traditional Chinese medicine compound treatment liquid group and positive drug control group were compared to analyze the regulatory effect of traditional Chinese medicine compound powder on pro-inflammatory factors induced by MS infection and the correlation between the concentration of traditional Chinese medicine compound treatment liquid and the expression level of key inflammatory factors. Key pro-inflammatory factors include tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), while key anti-inflammatory factors include interleukin-10 (IL-10). When the concentrations of key pro-inflammatory factor proteins in the model control group were higher than those in the blank control group, it indicated that MS infection induced pro-inflammatory phenomena. When the levels of key pro-inflammatory factor proteins decreased and the levels of key anti-inflammatory factor proteins recovered or increased in the traditional Chinese medicine compound treatment group compared to the model control group, it indicated that the traditional Chinese medicine compound treatment group could inhibit the excessive production of MS-induced pro-inflammatory factors. It also restored the expression of anti-inflammatory factors, thereby restoring immune homeostasis. Furthermore, with the increase of the concentration of the traditional Chinese medicine compound treatment liquid, the decrease in the protein level of key pro-inflammatory factors gradually increased compared with the model control group, indicating that the traditional Chinese medicine compound treatment liquid has a significant dose-dependent regulatory effect on MS infection-induced inflammatory response. By analyzing the expression level of key inflammatory factors mRNA, the expression level of key anti-inflammatory factors decreased and the expression level of key pro-inflammatory factors increased in the traditional Chinese medicine compound treatment liquid group, clarifying that the traditional Chinese medicine compound treatment liquid can inhibit the occurrence of excessive inflammation at both the transcriptional and translational levels.

[0021] One embodiment of the present invention is a method for verifying the regulation of a traditional Chinese medicine compound in treating Mycoplasma synoviae infection in chickens, including step four: detection of key proteins to clarify the regulatory effect of the traditional Chinese medicine compound powder on the recovery of the autophagy pathway; Key protein detection included collecting total cellular proteins from the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group after culture. Protein immunoblotting was used to detect the expression levels of key autophagy marker proteins LC3-Ⅰ, LC3-Ⅱ, Beclin-1, ATG5, ATG7, and p62. Autophagy flux was analyzed by comparing the LC3-Ⅱ / LC3-Ⅰ ratio and changes in p62 expression. The analysis of autophagy flux included an increase in the LC3-Ⅱ / LC3-Ⅰ ratio, a gradual recovery in the expression of Beclin-1, ATG5, and ATG7, and a decrease in p62 protein expression in the traditional Chinese medicine compound treatment liquid group. This indicated that the traditional Chinese medicine compound treatment liquid inhibited the overactivation of autophagy regulatory pathways and restored autophagy flux, enabling cells to promptly clear pathogens, damaged proteins, and damaged organelles, thereby reducing the inflammatory response. Based on genomics, autophagy and inflammation pathways were detected, and key targets in these pathways were identified using network diagrams. The PI3K-AKT-mTOR pathway was identified as the autophagy regulatory pathway, with key targets including AKT and mTOR. The NF-κB pathway was identified as the inflammation pathway, with key targets including IκBα and p65 proteins. Protein immunoblotting was used to detect the phosphorylation and expression levels of AKT and mTOR proteins in both pathways, as well as the phosphorylation and expression levels of IκBα and p65 proteins in the inflammation pathway. The regulatory effect of traditional Chinese medicine compound on the autophagy pathway was analyzed based on the detection results. The protein detection and analysis results included the following: Compared with the blank control group, the p-AKT and p-mTOR levels in the model control group were increased, as were the p-AKT / AKT and p-mTOR / mTOR ratios, p-IκBα / IκBα, and the expression level of nuclear NF-κBp65 protein. This indicated that the PI3K-AKT-mTOR pathway was overactivated, thereby inhibiting autophagic flux. Simultaneously, the inflammatory pathway was activated, inducing the overexpression of pro-inflammatory factors, leading to aggravated cellular inflammation. In the traditional Chinese medicine compound treatment group... Compared with the model control group, the p-AKT / AKT and p-mTOR / mTOR ratios of MS-infected cells decreased, the p-IκBα / IκBα ratio decreased, and the expression level of NF-κBp65 protein in the nucleus decreased. This indicates that the traditional Chinese medicine compound powder treatment can inhibit the autophagy pathway PI3K-AKT-mTOR and restore autophagic flux, while inhibiting the excessive inflammation mediated by NF-κB in the inflammatory pathway and restoring the expression of key anti-inflammatory factors. Thus, the traditional Chinese medicine compound powder can play a dual regulatory role in the treatment of Mycoplasma synoviae inflammation in chickens. Furthermore, for the TCM compound treatment groups with different concentrations of TCM compound treatment solution, cells and their supernatants were extracted at 6h, 12h, 24h, and 48h after the treatment solution was added. The transient expression levels of key inflammatory factors' mRNA, the LC3-II / LC3-I ratio, and changes in p62 expression, as well as the phosphorylation levels of IκBα and p65 proteins in the inflammatory pathway, were detected using protein immunoblotting. Autophagosome formation was observed under fluorescence microscopy in cells transfected with GFP-LC3 plasmid. The results were compared to analyze the sequential relationship between the recovery of autophagic flux, inhibition of the inflammatory pathway, and changes in key pro-inflammatory factors during MS-infected cell treatment with the TCM compound treatment solution. The results of the 6h cell and supernatant analysis compared to the model were also analyzed. In the control group, the LC3-II / LC3-I ratio increased, p62 expression level decreased, and phosphorylation levels of IκBα and p65 proteins decreased. The transient expression levels of key pro-inflammatory factor mRNAs began to decrease, and the results were obtained again at 12h and 24h. Compared with the results at 6h, the transient expression levels of key pro-inflammatory factor mRNAs, phosphorylation levels of IκBα and p65 proteins, and p62 expression levels further decreased, while the LC3-II / LC3-I ratio continued to increase. It can be seen that under the action of the traditional Chinese medicine compound dry powder treatment solution, the recovery of autophagic flux, inhibition of inflammatory pathways, and decrease in protein expression are time-related. That is, under the action of the traditional Chinese medicine compound dry powder treatment solution, the recovery of autophagic flux and the recovery of inflammatory pathways occur simultaneously, thereby more effectively achieving the purpose of inflammation treatment.

[0022] One embodiment of the present invention provides a method for verifying the regulation of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens, including step five: functional recovery experiment, to verify the regulatory effect of the traditional Chinese medicine compound powder on autophagic flux and the correlation between autophagy pathway and anti-inflammatory treatment; The functional recovery experiment included taking the successfully established MS infection cell model obtained in step one and randomly dividing them into an infection model control group, a traditional Chinese medicine compound treatment group, an autophagy inhibitor treatment group, and a traditional Chinese medicine compound treatment group combined with an autophagy inhibitor treatment group. The cells were cultured under the conditions in step two. Then, the cell viability and expression levels of key inflammatory factors in each experimental group were detected in steps two and three to verify the key molecular mechanism of traditional Chinese medicine compound in regulating MS infection inflammatory factors and autophagy flux recovery, and to clarify the association between autophagy pathway and MS infection inflammation. Comparative analysis of the data revealed that the cell survival rate in the traditional Chinese medicine compound treatment group was higher than that in the model control group, the expression levels of key pro-inflammatory factors were lower, and the expression levels of key anti-inflammatory factors were reduced. Simultaneously, the LC3-II / LC3-I ratio was increased and the p62 protein expression level was decreased, thus verifying that the traditional Chinese medicine compound treatment group can restore autophagic flux and exert anti-inflammatory effects. Furthermore, compared to the traditional Chinese medicine compound treatment group, the combined treatment group showed a lower LC3-II / LC3-I ratio, p62 protein re-accumulation, increased expression levels of key pro-inflammatory factor proteins, and a lower cell survival rate, thus verifying that the autophagy inhibitor reversed the therapeutic effect of the traditional Chinese medicine compound treatment group. Moreover, the traditional Chinese medicine compound powder restores autophagic flux by inhibiting the autophagy pathway, thereby achieving inflammation treatment and restoring immune homeostasis.

[0023] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for verifying the regulatory effects of a traditional Chinese medicine compound on Mycoplasma synoviae infection in chickens, used to study the therapeutic and regulatory effects of the traditional Chinese medicine compound on Mycoplasma synoviae infection in chickens, wherein the traditional Chinese medicine compound is a dry powder obtained by extraction, filtration, concentration, and freeze-drying of Astragalus membranaceus, Phellodendron chinense, Scutellaria baicalensis, Angelica sinensis, Chrysanthemum morifolium, Sophora japonica, Morus alba leaves, Ginkgo biloba leaves, and Taraxacum mongolicum, characterized in that: Includes the following steps: Step 1: Prepare the dry powder of traditional Chinese medicine compound and establish a cell model of Mycoplasma synoviae (MS) infection in chickens; Step 2: Determine the therapeutic effect of traditional Chinese medicine compound powder on MS-infected cells; Step 3: Detection of key inflammatory factors to clarify the inhibitory and regulatory effects of the traditional Chinese medicine compound powder on the excessive production of key pro-inflammatory factors and the restorative and enhancing regulatory effects on the expression of key anti-inflammatory factors; Step 4: Detection of key proteins to clarify the regulatory role of traditional Chinese medicine compound powder in the restoration of autophagy pathway; Step 5: Functional recovery experiment to verify the regulatory effect of traditional Chinese medicine compound powder on autophagic flux and the correlation between autophagy pathway and anti-inflammatory treatment.

2. The method for regulating and verifying the effects of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens according to claim 1, characterized in that: The establishment of the cell model of Mycoplasma synoviae infection in chickens includes: HD11 cells or primary chicken synovial cells were selected as cells to be infected. Cells in the logarithmic growth phase were seeded into multi-well culture plates and cultured at 37°C and 5% CO2 until they adhered to the plate. The culture system was then randomly divided into two groups: the infected system and the control system. The standard strain of Mycoplasma synoviae and an equal volume of cell culture medium were added to the infected system and the control system, respectively. The two systems were then cultured at 37°C and 5% CO2 to obtain the MS infection group and the control group. The establishment status of the MS infection cell model was detected by real-time PCR. The MS-specific nucleic acid and MS-specific DNA copy numbers in the two systems were detected. When the MS-specific nucleic acid detection result in the MS-infected group was positive, the MS-specific nucleic acid detection result in the judgment control group was negative, and the MS-specific DNA copy number in the MS-infected group was higher than that in the judgment control group, it indicated that the MS-infected group had successfully established a cell model of MS infection.

3. The method for regulating and verifying the effects of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens according to claim 2, characterized in that: The preparation of the traditional Chinese medicine compound dry powder includes, by weight percentage, the following traditional Chinese medicine raw materials: Astragalus membranaceus 10-20%, Phellodendron chinense 5-15%, Scutellaria baicalensis 8-15%, Angelica sinensis 5-12%, Chrysanthemum morifolium 8-15%, Sophora japonica 6-15%, Morus alba 8-15%, Ginkgo biloba 5-12%, and Taraxacum mongolicum 8-15%; The raw materials of traditional Chinese medicine were crushed and mixed separately. Then, the crude extract of the Chinese medicine was obtained by decoction or alcohol extraction. The crude extract was filtered or rotary evaporated to remove the alcohol solution, and the second extract was obtained. The second extract was concentrated and dried to obtain the compound powder of traditional Chinese medicine. The compound powder of traditional Chinese medicine was stored at -20℃ in the dark.

4. The method for regulating and verifying the effects of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens according to claim 3, characterized in that: The evaluation of the therapeutic effect of the traditional Chinese medicine compound dry powder on MS-infected cells includes mixing the traditional Chinese medicine compound dry powder with DMSO to prepare a treatment stock solution, and then adding cell culture medium to the treatment stock solution for dilution to obtain traditional Chinese medicine compound treatment solutions with concentrations of 25 μg / mL, 50 μg / mL and 100 μg / mL. For the successfully established MS infection cell model obtained in step one, they were randomly divided into a model control group, a traditional Chinese medicine compound treatment liquid group, and a positive drug control group. The judgment control group in step one was used as the blank control group in this step. 25 μg / mL, 50 μg / mL and 100 μg / mL of traditional Chinese medicine compound treatment liquid were added to the traditional Chinese medicine compound treatment liquid group and tylosin was added to the positive drug control group and cultured for 24 h. Cell viability in the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group after culture was detected by CCK-8 or MTT assay. Cell viability in the blank control group was used as the control standard and judgment criteria were set. Cell viability in the traditional Chinese medicine compound treatment liquid group was compared with the judgment criteria to obtain the concentration of non-cytotoxic traditional Chinese medicine compound treatment liquid. Supernatants from the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group were collected after culture. The 16S rRNA gene copy number of MS in the supernatant of each group was detected by real-time quantitative PCR. Combined with the cell viability detection results, the therapeutic effect of traditional Chinese medicine compound powder on MS-infected cells was clarified.

5. The method for regulating and verifying the effects of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens according to claim 4, characterized in that: The key inflammatory factors include key pro-inflammatory factors and anti-inflammatory factors; The detection of key inflammatory factors includes collecting cells and their supernatants from the cultured blank control group, model control group, traditional Chinese medicine compound treatment liquid group and positive drug control group, and using enzyme-linked immunosorbent assay (ELISA) to detect the protein secretion levels of key pro-inflammatory and anti-inflammatory factors in the supernatant. Total RNA was extracted from cells and their supernatants in each experimental group. The relative expression levels of key inflammatory factors mRNA in cells were detected by real-time quantitative PCR. The detection results of the blank control group, model control group, traditional Chinese medicine compound treatment liquid group and positive drug control group were compared to analyze the regulatory effect of traditional Chinese medicine compound powder on pro-inflammatory factors induced by MS infection and the correlation between the concentration of traditional Chinese medicine compound treatment liquid and the expression level of key inflammatory factors.

6. The method for regulating and verifying the effects of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens according to claim 5, characterized in that: The key protein detection included collecting total cellular proteins from the blank control group, model control group, traditional Chinese medicine compound treatment liquid group, and positive drug control group after culture. The expression levels of key autophagy marker proteins LC3-Ⅰ, LC3-Ⅱ, Beclin-1, ATG5, ATG7, and p62 were detected by protein immunoblotting. The autophagy flux was analyzed by comparing the LC3-Ⅱ / LC3-Ⅰ ratio and changes in p62 expression. Based on the detection of autophagy regulatory pathway and inflammation pathway using genomics, protein immunoblotting was used to detect the phosphorylation and expression levels of AKT and mTOR proteins in the autophagy regulatory pathway and inflammation pathway, as well as the expression levels of IκBα and p65 proteins in the inflammation pathway. The regulatory effect of traditional Chinese medicine compound treatment liquid on autophagy pathway was analyzed based on the detection results.

7. The method for regulating and verifying the effects of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens according to claim 6, characterized in that: The analysis of autophagic flux status included the observation that when the LC3-II / LC3-I ratio increased and the p62 protein expression level decreased in the traditional Chinese medicine compound treatment liquid group, it indicated that the traditional Chinese medicine compound treatment liquid inhibited the overactivation of autophagy regulatory pathways and restored autophagic flux status.

8. The method for regulating and verifying the effects of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens according to claim 7, characterized in that: The detection of key inflammatory factors and key proteins also includes collecting cells and their supernatants from the following groups at 6h, 12h, 24h, and 48h of culture: the group treated with traditional Chinese medicine compound therapy, the positive drug control group treated with tylosin, the blank control group, and the model control group. The transient changes in the relative expression levels of key inflammatory factor mRNAs are detected in step three, and the autophagic flux status and the phosphorylation level of proteins in the inflammatory pathway are detected in step four.

9. The method for regulating and verifying the effects of a traditional Chinese medicine compound for treating Mycoplasma synoviae infection in chickens according to claim 8, characterized in that: The functional recovery experiment included taking the successfully established MS infection cell model obtained in step one and randomly dividing it into an infection model control group, a traditional Chinese medicine compound treatment group, an autophagy inhibitor treatment group, and a combination of traditional Chinese medicine compound treatment and autophagy inhibitor treatment group. The cells were cultured under the conditions in step two. Then, the cell viability and expression levels of key inflammatory factors in each experimental group were detected in steps two and three to verify the key molecular mechanism of traditional Chinese medicine compound in regulating MS infection inflammatory factors and autophagy flux recovery, and to clarify the association between autophagy pathway and MS infection inflammation.