Application of AECII-sourced exosome miRNA in diagnosis and treatment of mycoplasma gallisepticum

Abstract

The invention belongs to the technical field of biology, and discloses a method for obtaining a miRNA (micro Ribonucleic Acid) of a cell exosome of a chicken alveolar type II epithelial cell AECII and application of the miRNA to treatment of Mycoplasma gallisepticum (MG) of the chicken alveolar type II epithelial cell AECII. The invention discloses the application of the exosome gga-miR-206 secreted by AECII cells in diagnosis and treatment of MG for the first time, provides a target spot of the gga-miR-206 for treating MG, and detects a potential mechanism of the gga-miR-206. It is found that the gga-miR-206 can inhibit proliferation of MG and relieve inflammatory response, and can be applied to treatment of mycoplasma gallisepticum. According to the invention, MG proliferation and gga-miR-206 expression of infected chick embryo lung tissues and correlation between the MG proliferation and the gga-miR-206 expression are also detected, so that the gga-miR-206 can be used as a biomarker for MG diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine and molecular biology, and in particular to an application of exosome miRNA derived from AECII in the diagnosis and treatment of Mycoplasma gallisepticum. Background Art

[0002] Mycoplasma gallisepticum (MG) is a type of mycoplasma. It is a highly pathogenic infectious pathogen that seriously affects the production traits and immune performance of poultry, and mainly causes chronic respiratory diseases in poultry. MG infection reduces the productivity of the poultry farming industry by 10% to 20% and increases the embryonic mortality rate by 5% to 10%. After poultry is infected with MG, it spreads rapidly and circulates continuously in the flock, seriously reducing the resistance of the flock, causing the flock to be infected with other pathogens such as Newcastle disease virus and Escherichia coli, further causing greater losses. The tissue lesions caused by MG infection of the host are mainly inflammatory lesions of the lungs such as inflammatory cell infiltration, alveolar congestion, and alveolar wall thickening. At present, the "gold standard" for the detection of MG mainly includes pathogen isolation and serological detection. However, these methods have high requirements for personnel and operation and are not suitable for rapid clinical diagnosis. In addition, the global prevention and control measures for MG mainly use tetracycline or macrolide antibiotics, but with the increasing problems of antibiotic resistance and drug residues, it poses a serious threat to the safety of poultry products. Therefore, it is necessary to find new rapid and accurate MG identification methods and new safe and effective treatment methods.

[0003] In recent years, exosomal miRNA is becoming a new regulator of cell function. As a communication medium, exosome-derived miRNA is easy to detect, not easy to degrade, biologically stable, and has the ability to interact with multiple target genes. Different cells can secrete or receive miRNA to regulate immune function. A large number of studies have shown that it can be used to diagnose and treat a variety of diseases.

[0004] miR-206 has been reported not only as a marker of T cell Th17-type immune response, but also as a tumor suppressor in various cancers such as colon cancer and lung cancer. Exosomal miR-206 has also been reported as a marker of hepatitis C virus infection and viral proliferation. However, to date, no studies have reported that miR-206 can indicate and treat MG infection.

[0005] References

[0006] 1.J.Beaudet,E.R.Tulman,K.Pflaum,X.Liao,G.F.Kutish,S.M.Szezepanek,L.K.Silbart,S.J.Geary,Transcriptional Profiling of the Chicken TrachealResponse to Virulent Mycoplasma gallisepticum Strain R(low),Infect Immun 85(10)(2017)

[0007] 2.Yadav JP,Batra K,Singh Y,Singh M.Comparative evaluation of indirectELISA and DOT blot assay for serodetection of Myeoplasma gallisepticum andMycoplasma synoviae antibodies in poultry.J Microbiol Methods.2021 Oct;189:106317.

[0008] 3.Bartel DP.MicroRNAs:genomics,biogenesis,mechanism,andfunction.TroCell.2004 Jan 23;116(2):281-97.

[0009] 4.Ye X,Lu Q,Yang A,Rao J,Xie W,He C,Wang W,Li H,Zhang Z.MiR-206regulates the Th17 / Treg ratio during osteoarthritis.Mol Med.2021 Jun 19;27(1):64.

[0010] 5.Nguyen Thi Nhung,Niwat Chansiripornchai,Juan J Carrique-Mas.Antimicrobial Resistance in Bacterial Poultry Pathogens:A Review.FrontVet Sci.2017 Aug 10:4:126.

[0011] 6.Liao TL, Chen YM, Hsieh SL, Tang KT, Chen DY, Yang YY, Liu HJ, YangSS.Hepatitis C Virus-Induced Exosomal MicroRNAs and Toll-Like Receptor

[0012] 7.Polymorphism Regulate B-Cell Activating Factor.mBio.2021 Dec 21;12(6):e0276421. Summary of the invention

[0013] The first object of the present invention is to provide a safe and effective strategy for treating MG infection by using exosomal gga-miR-20 derived from AECII.

[0014] The second purpose of this study is to provide the application of gga-miR-206 as a biomarker for MG infection.

[0015] The technical solution adopted by the present invention is:

[0016] Application of AECII-derived exosomal miRNA in the treatment of MG.

[0017] Step 1: Alveolar epithelial type II cells (AECII) were isolated and purified from SPF chicken embryos, and the purified AECII-derived exosomes were extracted and compared with the negative group. MG infected AECII cells and promoted the expression of gga-miR-206 in exosomes.

[0018] Step 2: gga-miR-206 is delivered from AECII cells to HD11 cells via exosomes.

[0019] Step 3: gga-miR-206 can inhibit the proliferation and adhesion of MG in HD11 cells.

[0020] Another object of the present invention is the use of exosomal miRNA derived from AECII in the diagnosis of MG.

[0021] The expression of gga-miR-206 in MG-infected chicken embryos showed a significant negative correlation with the copy number of Mycoplasma gallisepticum in chicken embryos infected with MG.

[0022] The beneficial effects of the present invention are:

[0023] Exosomes from AECII rich in gga-miR-206 are used to resist MG infection, significantly inhibit the proliferation and adhesion of MG, improve the hatching rate of chicken embryos, and alleviate lung tissue damage. While enhancing the therapeutic effect, it greatly reduces the problems of drug resistance and drug residue caused by the abuse of antibiotics. Gga-miR-206 can be synthesized artificially, with low cost, convenient storage, fast onset, simple operation, and will not cause side effects such as drug resistance and drug residue. It can be used in large-scale production and application, and has good clinical application value and broad application prospects. At the same time, it can also be used as a biomarker for detecting MG infection, and quickly and accurately realize the early diagnosis and rapid control of MG. In order to more clearly explain the characteristics and effects of the present invention, the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Figure A shows the exosomes secreted by AECII identified by transmission electron microscopy. Figure 1 Figure B shows the size distribution of exosomes secreted by AECII. Figure 1 Figure C is the identification diagram of the surface protein markers of exosomes secreted by AECII. Figure 1 Figure D shows the expression level of gga-miR-206 in exosomes secreted by AECII.

[0025] Figure 2 Figure A shows the status of AECII cells in the negative control group. Figure 2 Figure B shows the transfection efficiency test of CY3-gga-miR-206. Figure 2 Figure C shows gga-miR-206 delivered to HD11 cells via exosomes.

[0026] Figure 3 Figure A shows the changes in MG copy number after HD11 was treated with MG, C-sEV, MG-sEV, and gga-miR-206 mimics. Figure 3 Figure B shows the expression of MG adhesion protein pMGA1.2 in HD11 cells after treatment with MG, C-sEV, MG-sEV, and gga-miR-206 mimics. Figure 3 Figure C shows the detection of MG invasion into HD11 cells by C-sEV, MG-sEV, and gga-miR-206 mimics.

[0027] Figure 4 Figure A shows the changes in MG copy number in chicken embryos infected with MG on different days. Figure 4 Figure B shows the expression of gga-miR-206 in chicken embryos infected with MG on different days. Figure 4 Figure C shows the correlation between MG copy number and gga-miR-206 expression in chicken embryos infected with MG on different days. Figure 5 Flow chart for chick embryo injection. Figure 6 Hatching rate of chicken embryos 19-21 days after HD11 treatment with MG, C-sEV, MG-sEV, and gga-miR-206 mimics. Figure 7 Figure A shows the MG copy number in lung tissue of chicks hatched after treatment with MG, C-sEV, MG-sEV, and gga-miR-206 mimics. Figure 7 Figure B shows the expression of adhesion protein pMGA1.2 in lung tissue of chicks hatched after treatment with MG, C-sEV, MG-sEV, and gga-miR-206 mimics. Figure 8 The degree of damage to various organs in hatched chicks was observed after treatment with MG, C-sEV, MG-sEV, and gga-miR-206 mimics. Fig. 9 HE-stained sections of lung tissues of hatched chicks after treatment with MG, C-sEV, MG-sEV, and gga-miR-206 mimics. Fig.10 These are the immune organ indexes of the thymus, spleen and bursa of Fabricius in hatched chicks after treatment with MG, C-sEV, MG-sEV and gga-miR-206 mimics. Fig.11 Detection of inflammatory factors and inflammasome proteins in lung tissue of hatched chicks after treatment with MG, C-sEV, MG-sEV, and gga-miR-206 mimics. DETAILED DESCRIPTION

[0028] The inventors established a chicken embryo MG infection model using microinjection technology

[0029] 1. The experimental groups were negative control group, MG infection group, C-sEV treatment group, MG-sEV treatment group, agomiR-206 injection group and agomiR-206-NC injection group.

[0030] 2. The specific experimental process is as follows: MG infection was performed on the 13th day of incubation in the incubator, and C-sEV, MG-sEV, agomiR-206 and agomiR-206-NC were treated on the 15th and 17th days of incubation, respectively. The negative control group and MG group were subjected to the same operation with normal saline. Figure 5 shown.

[0031] 3. Hatching rate statistics: The hatching rate of the chicken embryos in each treatment group was recorded from the 19th day of incubation. The incubation of the chicken embryos continued until the 21st day. Figure 6 shown.

[0032] 4. MG proliferation and gga-miR-206 expression detection: The copy number of MG and the expression of adhesion protein pMGA1.2 and gga-miR-206 were detected using lung tissue. Figure 7 shown.

[0033] 5. Observation of organ damage: After hatching, the trachea, heart, liver, spleen, lung, kidney and bursa of Fabricius were dissected and photographed to compare the effects of different treatments on organ damage, such as Figure 8 In addition, the lung lesions were observed by HE staining, as shown in Fig. 9 shown.

[0034] 6. Immune index statistics: After hatching, the thymus, spleen and bursa of Fabricius are weighed. Statistical immune organ index, such as Fig.10 shown.

[0035] 7. Inflammatory response detection: Detect the expression of inflammatory factors and inflammasomes in lung tissue, such as Fig.11 shown.

[0036] By comparing with agomiR-206-NC, C-sEV and MG infection groups, it can be proved that MG-sEV and agomiR-206 can significantly inhibit organ enlargement, inhibit MG replication, and reduce the congestion of the alveolar cavity, blurred alveolar cavity outline and thickening of alveolar wall in chickens caused by MG. In addition, MG-sEV and agomiR-206 can significantly reduce the expression of pro-inflammatory factors TNF-α and IL-18 and inflammasome-related proteins NLRP3, IL-1β and ASC in the lungs of chickens infected with MG. This shows that MG-sEV and agomiR-206 can reduce the lung damage and inflammatory response of chickens caused by MG infection, and have a significant protective effect.

[0037] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not equivalent to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. An application of exosomal miRNA derived from AECII in the diagnosis and treatment of Mycoplasma gallisepticum, characterized in that: The exosomal miRNA is gga-miR-206.

2. The use of an AECII-derived exosomal miRNA for the diagnosis and treatment of Mycoplasma gallisepticum according to claim 1, wherein the AECII exosomal miRNA is isolated and obtained from chicken embryo primary type II epithelial cells.

3. A method for obtaining exosome miRNA in the application of an AECII-derived exosome miRNA in the diagnosis and treatment of Mycoplasma gallisepticum according to any one of claims 1-2, characterized in that: The specific steps are: Step 1: Use collagenase lysis method to separate and purify AECII cells from lung tissue of 14-day-old SPF chicken embryos under sterile environment. Step 2: Infect AECII cells with Mycoplasma gallisepticum, and use gradient centrifugation to obtain exosomes from AECII cells in the blank group and the infected group, respectively.

4. qPCR detection of the use of an AECII-derived exosome miRNA as claimed in claim 3 for the diagnosis and treatment of Mycoplasma gallisepticum, characterized in that: Differential expression of gga-miR-206 in the blank group and the Mycoplasma gallisepticum infection group.

5. The use of an exosomal miRNA derived from AECII as claimed in any one of claims 1 to 3 for the diagnosis and treatment of Mycoplasma gallisepticum, characterized in that: The specific steps are: Step 1: Establishment of the chicken macrophage HD11 cell model in which gga-miR-206 inhibits Mycoplasma gallisepticum infection; Step 2: Detection of the inhibitory effect of gga-miR-206 on the proliferation and adhesion of Mycoplasma gallisepticum; Step 3. The expression of gga-miR-206 in chicken embryos infected with Mycoplasma gallisepticum was negatively correlated with the copy number of Mycoplasma gallisepticum.

6. The use of an AECII-derived exosomal miRNA for the diagnosis and treatment of Mycoplasma gallisepticum as claimed in claim 5, characterized in that: The step one comprises: To determine whether gga-miR-206 can inhibit the proliferation of Mycoplasma gallisepticum in chicken macrophages HD11, we selected a virulent strain of Mycoplasma gallisepticum and measured it by the expression of Mycoplasma gallisepticum copy number and adhesion protein. The preparation of the cell model proved that gga-miR-206 could inhibit the proliferation of Mycoplasma gallisepticum through the negative control group, exosome incubation group and gga-miR-206 mimic treatment group.

7. The use of an AECII-derived exosomal miRNA for the diagnosis and treatment of Mycoplasma gallisepticum as claimed in claim 5, characterized in that: The second step comprises: (1) To determine whether gga-miR-206 can inhibit the proliferation of Mycoplasma gallisepticum, the cells were divided into a negative control group, an exosome incubation group, and a gga-miR-206 mimic treatment group. (2) To determine whether gga-miR-206 can inhibit the adhesion of M. gallisepticum, the cells were divided into a negative control group, an exosome incubation group, and a gga-miR-206 mimic treatment group, which was measured by the fluorescence expression of the nuclei of M. gallisepticum-invaded cells.

8. The use of an AECII-derived exosomal miRNA for the diagnosis and treatment of Mycoplasma gallisepticum as claimed in claim 5, characterized in that: The step three comprises: (1) Determine the negative correlation between the expression level of gga-miR-206 and the copy number of Mycoplasma gallisepticum in chicken embryos infected with Mycoplasma gallisepticum; (2) The expression level of gga-miR-206 in lung tissue and the copy number of Mycoplasma gallisepticum on days 3, 4, 6, 8, 9, 10 and 11 of chicken embryos infected with Mycoplasma gallisepticum proved that there was a significant correlation between the copy number of Mycoplasma gallisepticum and the expression level of gga-miR-206 during the infection of chicken embryos with Mycoplasma gallisepticum.

Images