Traditional Chinese medicine composition for treating bursa of Fabricius immunosuppression of Lingnan yellow chicken

Abstract

The invention belongs to the technical field of poultry immunosuppression, and discloses a traditional Chinese medicine composition for treating bursa of Fabricius immunosuppression of Lingnan yellow chicken, which is prepared from the following raw materials in parts by weight: 80-100 parts of rhizoma atractylodis macrocephalae fried with bran, 50-70 parts of codonopsis pilosula, 50-70 parts of dandelion, 40-60 parts of poria cocos, 20-40 parts of rhizoma cyperi, 10-30 parts of pericarpium citri reticulatae, 10-30 parts of fructus amomi, 20-40 parts of liquorice, 10-30 parts of hawthorn, 10-30 parts of medicated leaven and 10-30 parts of malt. 10 to 30 parts of fossil fragments and 10 to 30 parts of oysters; the traditional Chinese medicine composition increases the volume and index of bursa of Fabricius of Lingnan yellow chickens, promotes the development of bursa of Fabricius, relieves the tissue structure damage of bursa of Fabricius, and can treat the immunosuppression of bursa of Fabricius.

Description

Technical Field

[0001] The invention relates to the technical field of treating poultry immunosuppression, in particular to a traditional Chinese medicine composition for treating bursal immunosuppression of Lingnan yellow chicken. Background Art

[0002] At present, most of the poultry farming in my country is based on free-range farming by farmers, with a low degree of intensiveness, uneven breeding levels among farmers, and a lack of relevant feeding management and disease prevention knowledge, which leads to a relatively high incidence and recurrence rate of some diseases. In particular, some immunosuppressive diseases are extremely harmful to the breeding industry. They can reduce the immunity and resistance of poultry, reduce the ability to respond to vaccines and diseases, significantly increase the incidence rate, make disease treatment difficult, and general drugs are difficult to achieve ideal results. In addition, the feed-to-meat ratio and feed-to-egg ratio are increased, making breeding difficult and the breeding cost high.

[0003] The main immune mode of poultry is cellular immunity. As a unique immune organ of poultry, the bursa of Fabricius can not only provide the necessary microenvironment for the development and maturation of B cells as a primary immune organ, but also participate in the body's immune response as a secondary immune organ. Therefore, promoting the development of the bursa of Fabricius and preventing it from being damaged are of great significance for improving the immunity of poultry. However, there is still a lack of Chinese herbal compound that can effectively promote the development of the bursa of Fabricius and treat bursa of Fabricius immunosuppression on the market. Summary of the invention

[0004] The present invention aims to provide a traditional Chinese medicine composition for treating immunosuppression of the bursa of Fabricius in Lingnan yellow chicken, which can increase the volume and index of the bursa of Fabricius in Lingnan yellow chicken, promote the development of the bursa of Fabricius, alleviate the damage of the bursa tissue structure, and treat the immunosuppression of the bursa of Fabricius.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A traditional Chinese medicine composition for treating immunosuppression of bursa of Fabricius in Lingnan yellow chicken is prepared from the following raw materials in parts by weight: 80-100 parts of bran-fried atractylodes macrocephala, 50-70 parts of codonopsis pilosula, 50-70 parts of dandelion, 40-60 parts of tuckahoe, 20-40 parts of cyperus rotundus, 10-30 parts of tangerine peel, 10-30 parts of amomum villosum, 20-40 parts of liquorice, 10-30 parts of hawthorn, 10-30 parts of Shenqu, 10-30 parts of malt, 10-30 parts of dragon bone and 10-30 parts of oyster.

[0007] Furthermore, it is made from the following raw materials in parts by weight: 90 parts of fried Atractylodes macrocephala with bran, 60 parts of Codonopsis pilosula, 60 parts of Taraxacum herb, 50 parts of Poria cocos, 30 parts of Cyperus rotundus, 20 parts of dried tangerine peel, 20 parts of Amomum villosum, 30 parts of Licorice, 20 parts of Hawthorn, 20 parts of Shenqu, 20 parts of malt, 20 parts of Dragon Bone, and 20 parts of Oyster.

[0008] The beneficial effects of the technical solution are:

[0009] The Chinese medicinal composition for treating immunosuppression of bursa of Fabricius of Lingnan yellow chicken can increase the volume and index of bursa of Fabricius of Lingnan yellow chicken and promote the development of bursa of Fabricius; and can make the tissue structure of immunosuppressed bursa of Fabricius normal, with clear follicular structure, orderly distribution, close arrangement of cells, clear boundary between cortex and medulla, rich lymphocytes in medulla area, and sparse reticular structure, so as to treat immunosuppressive diseases of bursa of Fabricius; and the Chinese medicinal composition has a significant regulating effect on the expression of key molecules in mitogen activated protein kinase (MAPK) signal pathway in the process of relieving immunosuppression of bursa of Fabricius of Lingnan yellow chicken; therefore, the medicine of the present invention will play an important role in preventing and treating immunosuppressive diseases of avian animals, can greatly improve the resistance of the body to diseases, reduce the huge losses caused by immunosuppressive diseases to the breeding industry, and has a very significant therapeutic effect; meanwhile, it is easy to use, has low cost, can be directly mixed in chicken feed for consumption, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is a bar graph of body weight, bursal weight, and bursal organ index of the control group, TCM group, TCM+CTX group, and CTX group in Example 1 of the present invention;

[0011] Figure 2 The results of bursal histological observation (100×; 200×; 400×) of the control group, TCM group, TCM+CTX group and CTX group in Example 1 of the present invention are shown;

[0012] Figure 3 It is a bar graph of the total area of ​​bursa vesicles, cortical area, medullary area, and the ratio of cortical area to medullary area in the control group, TCM group, TCM+CTX group, and CTX group in Example 1 of the present invention;

[0013] Figure 4 The distribution diagram of DEGs in the bursa of Lingnan yellow chicken in the CTX group and the CTX+TCM group in Example 2 of the present invention; in the figure, (a) is a volcano map of DEGs in the CTX group and the CTX+TCM group; (b) is a heat map of DEGs in the CTX group and the CTX+TCM group; (c) is a GO bar graph of DEGs in the CTX group and the CTX+TCM group; (d) is a KEGG bubble map of DEGs in the CTX group and the CTX+TCM group;

[0014] Figure 5 This is a schematic diagram of the interaction of differentially expressed gene proteins in Example 2 of the present invention;

[0015] Figure 6 The expression level of key genes in the MAPK signaling pathway in Example 2 of the present invention is shown in FIG. Figure 1 ;

[0016] Figure 7 The expression level of key genes in the MAPK signaling pathway in Example 2 of the present invention is shown in FIG. Figure 2 ;

[0017] Figure 8 This is a bar graph of protein concentrations in the MAPK signaling pathway in Example 2 of the present invention. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments:

[0019] A traditional Chinese medicine composition for treating immunosuppression of bursa of Fabricius in Lingnan yellow chicken is prepared from the following raw materials in parts by weight: 90g of bran-fried atractylodes macrocephala, 60g of codonopsis pilosula, 60g of dandelion, 50g of tuckahoe, 30g of cyperus rotundus, 20g of tangerine peel, 20g of amomum villosum, 30g of liquorice, 20g of hawthorn, 20g of Shenqu, 20g of malt, 20g of dragon bone, and 20g of oyster.

[0020] The preparation process of the above-mentioned Chinese medicine composition is as follows:

[0021] Step 1: weigh 90g of bran-fried Atractylodes macrocephala, 60g of Codonopsis pilosula, 60g of Taraxacum mongolicum, 50g of Poria cocos, 30g of Cyperus rotundus, 20g of dried tangerine peel, 20g of Amomum villosum, 30g of Licorice root, 20g of Crataegus pinnatifida, 20g of Shenqu, 20g of Malt, 20g of Dragon Bone, and 20g of Oyster, and crush all the drugs into foam;

[0022] Step 2: Put the crushed medicine powder into a casserole, add water three times the weight of the medicine into the casserole, soak for 30 minutes and then start boiling. After boiling over high heat, simmer over low heat for 30 minutes. Filter through gauze to obtain the decoction, and then mix the decoction evenly into the basic feed.

[0023] The following experiment was conducted using the feed prepared in step 4 above.

[0024] Example 1

[0025] (1) Experimental animals and sample preparation

[0026] In this experiment, 240 one-day-old Lingnan yellow chickens, half male and half female, were randomly divided into 4 groups, with 6 replicates in each group and 10 chickens in each replicate. There was no significant difference in body weight between replicates after statistical analysis (P>0.05). The control group and cyclophosphamide group (CTX group) were fed with a basal diet, while the Chinese medicine group (TCM group) and Chinese medicine + cyclophosphamide group (TCM + CTX group) added 400 mg / kg of Chinese medicine formula to the basal diet. During the experiment, all groups maintained the same diet. At 20, 21, and 22 days of age, the CTX group and TCM + CTX group received a leg muscle injection of 0.5 mL of cyclophosphamide (20 mg / kg), while the control group and TCM group received an equal amount of saline injection. The experiment lasted for 27 days, and the chickens were fasted for 12 hours before feeding. Serum and bursa of Fabricius were then collected and processed and stored according to the established protocol for subsequent analysis.

[0027] (2) Bursal organ index test

[0028] After collecting the complete bursal sample, it was immediately rinsed with saline for 2-3 times, dried with filter paper and weighed. The bursal organ index was calculated based on the body weight before collection and the bursal weight. The calculation formula was: bursal organ index = bursal weight (g) / body weight (kg).

[0029] (3) Observation of bursal tissue morphology

[0030] Fresh bursal tissues were fixed in 4% paraformaldehyde for 48 hours, then embedded in paraffin, sliced ​​(5 μm thick), and stained with hematoxylin-eosin (HE). The bursal tissue structure was observed using an optical microscope and images were collected using CaseViewer software at 200x and 400x magnifications.

[0031] (4) RNA extraction and library construction

[0032] Six long RNA libraries were extracted from the bursa of Lingnan yellow chicken, including biological samples from the cyclophosphamide group and the Chinese medicine + cyclophosphamide group, with 3 samples in each group (n=3). Total RNA was extracted and purified using Trizol reagent, and quality assessment was performed using NanoDrop ND-1000 UV-Vis spectrophotometer and Bioanalyzer 2100 to ensure that the RNA concentration was greater than 50 ng / μL, the RNA integrity number (RIN) was greater than 7.0, and the optical density ratio (OD 260 / 280) was greater than 1.8. RNA from three chickens in each group was used for RNA sequencing, and 10 μg of total RNA was treated with the Ribo-Zero Gold rRNA Removal Kit. After RNA fragmentation, reverse transcription, ligation, and PCR amplification, cDNA libraries were prepared and paired-end sequencing was performed on the Illumina HiSeq 4000 platform by Lianchuan Biotechnology Co., Ltd. In addition, small RNA libraries were constructed using an RNA sample preparation kit and sequenced on an Illumina HiSeq 2000 / 2500 platform with a read length of 50 bases.

[0033] (5) DEGs identification and functional enrichment analysis

[0034] After sequencing, data filtering and validation were performed using Cutadapt (V 4.2) and FastQC (V 4.2) software to ensure high-quality, clean datasets. Subsequently, the dataset was aligned to the chicken reference genome (GCF_002166845.1) using HISAT2 (V 2.2.1) software. Transcripts were assembled from the aligned reads of each sample using StringTie (V 2.1.6) software, and the composite transcriptome was subsequently reconstructed using gffcompare (V 0.9.8) software. The expression levels of transcripts were evaluated using StringTie (V 2.1.6) and ballgown (V 2.30.0) software, and the FPKM values ​​were calculated. The identification criteria for DEGs were a P value less than 0.05 and an absolute log2-fold change greater than 2. For DEG analysis, Gene Ontology (GO) classification was performed through the GO website (http: / / geneontology.org / ), and KEGG pathway analysis was performed on the same DEGs using the KOBAS web server (http: / / kobas.cbi.pku.edu.cn / ). GO terms and KEGG pathways with P values ​​less than 0.05 were considered significantly enriched.

[0035] (6) Protein-protein interaction (PPI) network analysis

[0036] The STRING 10 database (http: / / string-db.org / ) was used as a query tool to determine the protein interaction relationship between differentially expressed genes. At the same time, the enriched genes in the key pathways screened in this experiment were integrated into the STRING database. The analysis results were visualized using Cytoscape_v3.2.1 software. In this PPI network, each point represents a biomolecule, the lines represent the interactions between biomolecules, and the size of the point is adjusted according to the number of lines it connects: the more lines connected, the larger the size of the point.

[0037] (7) qRT-PCR detection

[0038] The total RNA of each sample was reverse transcribed into cDNA using the TaKaRa Reverse Transcription Kit (TaKaRa Bio Inc., Shiga, Japan). The reaction mixture consisted of 10 μL SYBR Green Master Mix, 7 μL RNase-Free dH2O, 1 μL each of forward and reverse primers, and 1 μL each of cDNA, with a final volume of 20 μL. The thermal cycling protocol began with a pre-denaturation step at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s for denaturation, 60°C for 30 s for annealing, and 72°C for 30 s for extension. actb was used as an internal reference gene to normalize the expression level. 2 -ΔΔCT The formula calculates the relative mRNA expression level of the target gene to allow quantitative comparison of gene expression between samples.

[0039] (8) Western blot detection method

[0040] Total protein was extracted from the bursa tissue of Lingnan yellow chicken using RIPA lysis buffer (Beyotime, Shanghai, China), and the protein concentration of each group of samples was determined using a BCA protein concentration assay kit (Beyotime, Shanghai, China). Subsequently, 5 volumes of SDS-PAGE protein loading buffer were added to each sample, mixed thoroughly, and denatured. According to standard operating procedures, proteins were separated by electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes containing the target proteins were blocked in 5% skim milk powder and then incubated overnight with the following antibodies: GAPDH, HRAS, C-FOS, EREG (all purchased from Proteintech, Wuhan, China). The next day, secondary antibodies were used for incubation for 1 hour, and then the color development reaction was completed. The protein expression on the PVDF membrane was detected using a fully automatic chemiluminescence imaging system, and quantitative analysis was performed using Image J software.

[0041] (9) Statistical data analysis

[0042] The data obtained in this experiment were statistically analyzed and tested for significance using SPSS26.0 software. The differences in parameters such as the bursal index and the ratio of the bursal cortex to the medulla area were evaluated by one-way analysis of variance (ANOVA). For the verification of RNA-Seq results and the identification of key genes, a two-tailed t-test was used for statistical evaluation. The graphical display of the experimental results was completed using GraphPad Prism 5.0 software. The data were presented as mean ± standard error (SEM), and a P value less than 0.05 was used as the criterion for statistical significance.

[0043] From the above experimental results, we can see that Figures 1 to 3 The figure is a graph showing the effect of the Chinese medicine composition of the present invention on the reduction of the bursal organ index and the tissue structure lesions of Lingnan yellow chicken induced by CTX, wherein Figure 1 It can be seen that compared with the control group, the bursa of the TCM group and the TCM+CTX group were similar in size, while the bursa of the CTX group was smaller. The organ index results showed that the bursa index of the CTX group was significantly lower than that of the control group, while the Chinese medicine composition significantly increased the bursa index. The above results show that the volume and index of the bursa of the Lingnan yellow chicken were significantly reduced under the induction of CTX, and the Chinese medicine composition can alleviate this phenomenon.

[0044] from Figure 2 It can be seen that the bursal tissue stained with hematoxylin-eosin (H&E) showed that the bursal follicles of the CTX group were disordered, the interfollicular space was narrow, and there were a large number of cell infiltrations; the density of lymphocytes in the cortical area was significantly reduced, and the reticular cells in the medulla area were significantly proliferated. In contrast, the bursal tissue structure of the control group, TCM group, and TCM+CTX group was normal, with clear follicular structure, orderly distribution, and densely arranged cells; the boundary between the cortex and medulla was clear, the medulla area was rich in lymphocytes, and the reticular structure was sparse.

[0045] also, Figure 3 It can be more intuitively shown that the cortical area of ​​the CTX group was significantly smaller than that of the control group and the TCM+CTX group, the medullary area was larger, and the ratio of the cortical to medullary area was significantly reduced (P<0.05). In short, under CTX induction, the bursal index, cortical to medullary area ratio and structural integrity of Lingnan yellow chicken were significantly reduced, and the Chinese medicine composition can alleviate this phenomenon.

[0046] In summary, the Chinese medicine composition of the present invention can increase the volume and index of the bursa of Fabricius of Lingnan yellow chicken and promote the development of the bursa of Fabricius; and can make the tissue structure of the immunosuppressed bursa of Fabricius normal, with clear follicular structure, orderly distribution, dense arrangement of cells, clear boundary between cortex and medulla, rich lymphocytes in the medulla area, and sparse reticular structure, thereby treating immunosuppressive diseases of the bursa of Fabricius.

[0047] Example 2

[0048] (1) Identification and functional enrichment analysis of differentially expressed genes (DEGs)

[0049] like Figure 4 The following is the distribution of DEGs in the bursa of Lingnan yellow chicken in the CTX group and the CTX+TCM group; the statistical thresholds P<0.05 and |log2(FC)>1| were used to analyze the transcriptome data of the bursa of Lingnan yellow chicken in the CTX group and the TCM+CTX group. A total of 1452 DEGs were successfully identified, including 208 down-regulated genes and 1244 up-regulated genes, such as Figure 4 (a) As shown. To further elucidate the mechanism by which the Chinese medicine composition alleviates CTX-induced immunosuppression, functional enrichment analysis of DEGs was performed in this experiment. In the comparison between the CTX group and the TCM+CTX group, a total of 45 gene ontology (GO) terms were enriched, including 20 biological process (BP) terms, 15 cellular component (CC) terms, and 10 molecular function (MF) terms, as shown in Figure 2. Figure 4 (c) The results of KEGG enrichment analysis are shown in Figure 4 As shown in (d), these DEGs were mainly enriched in signaling pathways such as MAPK signaling pathway, calcium signaling pathway, and Wnt signaling pathway (P<0.05). From the enriched KEGG pathways, it was observed that the MAPK signaling pathway contained a relatively large number of DEGs, and these DEGs showed a consistent trend.

[0050] (2) Protein-protein interaction (PPI) network

[0051] In this experiment, the protein-protein interaction (PPI) network of all differentially expressed genes (DEGs) was constructed using the STRING 10 database, and their possible interaction patterns were further analyzed. The results showed that a total of 217 DEGs were correlated, such as Figure 5 As shown. Among these 217 related DEGs, 32 DEGs were enriched in the MAPK signaling pathway. PPI network analysis confirmed that there were strong biological interaction relationships among EREG, C-FOS, JUN, NTRK2, MAPK10, etc. In addition, most of the DEGs related to the MAPK signaling pathway showed a down-regulation trend.

[0052] In summary, this experiment determined that the MAPK signaling pathway plays a key role in alleviating CTX-induced bursal immunosuppression in Lingnan yellow chickens through comprehensive analysis of the PPI network. Specifically, EREG, C-FOS, JNK, and hRAS occupy a core position in the MAPK signaling pathway. Therefore, this experiment used Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) techniques to evaluate the relative expression levels of EREG, C-FOS, JNK, and hRAS protein genes in the bursal tissues of Lingnan yellow chickens in the CTX group and TCM+CTX group, as well as the expression levels of other DEGs, such as Figures 6 to 8 The results showed that compared with the CTX group, the levels of EREG, C-FOS, JNK, and hRAS genes and proteins in the TCM+CTX group were significantly reduced, and the level of core protein phosphorylated JNK was also significantly reduced in the TCM+CTX group. This indicates that the Chinese medicine composition has a regulatory effect on the MAPK signaling pathway.

[0053] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A Chinese medicine composition for treating immunosuppression in bursa of Fabricius of Lingnan yellow chicken, characterized in that: The invention is prepared from the following raw materials in parts by weight: 80-100 parts of fried atractylodes macrocephala with bran, 50-70 parts of codonopsis pilosula, 50-70 parts of dandelion, 40-60 parts of tuckahoe, 20-40 parts of cyperus rotundus, 10-30 parts of tangerine peel, 10-30 parts of amomum villosum, 20-40 parts of liquorice, 10-30 parts of hawthorn, 10-30 parts of Shenqu, 10-30 parts of malt, 10-30 parts of dragon bone and 10-30 parts of oyster.

2. A Chinese medicine composition for treating immunosuppression in bursa of Fabricius of Lingnan yellow chicken according to claim 1, characterized in that: The invention is prepared from the following raw materials in parts by weight: 90 parts of fried Atractylodes macrocephala with bran, 60 parts of Codonopsis pilosula, 60 parts of Taraxacum herb, 50 parts of Poria cocos, 30 parts of Cyperus rotundus, 20 parts of dried tangerine peel, 20 parts of Amomum villosum, 30 parts of Licorice root, 20 parts of Crataegus pinnatifida, 20 parts of Shenqu, 20 parts of malt, 20 parts of Dragon bone and 20 parts of Oyster.

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