Application of tyrosine hydroxylase in medicine for treating collagen-induced arthritis

Abstract

The invention discloses application of tyrosine hydroxylase in drugs for treating collagen-induced arthritis, and belongs to the technical field of biological medicines, TH expression is down-regulated by adopting a TH-shRNA plasmid, TH expression is up-regulated by adopting a plasmid for expressing the full length of a mouse TH gene, and the total length of the mouse TH gene is up-regulated. The TH-shRNA plasmid and the plasmid for expressing the full length of the TH gene of the mouse are respectively transfected with a collagen induced arthritis (CIA) mouse CD4 + T cell and a CIA mouse CD4 + T cell, and the imbalance condition of Th17 / Treg in the CD4 + T cell is observed. The result shows that the TH gene silencing in the CD4 + T cell of the CIA can obviously promote the imbalance of the Th17 / Treg, the TH gene overexpression in the CD4 + T cell can obviously inhibit the imbalance of the Th17 / Treg, and the Plt can obviously inhibit the TH gene overexpression in the CD4 + T cell. And 0.05%.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of tyrosine hydroxylase in a drug for treating collagen-induced arthritis. Background Art

[0002] Catecholamines (CAs) mainly include norepinephrine, epinephrine and dopamine. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of CAs.

[0003] For a long time, CAs have been considered to be mainly synthesized and secreted by nerve cells and endocrine cells to regulate the body's circulation, respiration, digestion and other functions. Recent studies have found that CAs are also involved in regulating the body's immune function. Studies have found that CAs can regulate the differentiation and function of helper T (T helper, Th) cells and promote the differentiation of Th1 cells into Th2 cells, suggesting that CAs have the effect of inhibiting proinflammatory immune cells and that CAs also have an effect on autoimmune diseases. Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovial inflammation accompanied by articular cartilage and bone damage. The functional imbalance between Th17 cells and regulatory T (Treg) cells is one of the pathogenesis of RA. Using the animal model of RA, collagen-induced arthritis (CIA), this study explored the effect of CAs on the Th17 / Treg imbalance in CIA mice, providing new clues for the neuroendocrine regulatory mechanism of autoimmune diseases. Summary of the invention

[0004] Technical issues solved:

[0005] The present application aims to address the shortcomings of the prior art, such as the lack of reports on the use of tyrosine hydroxylase in the treatment of collagen-induced arthritis, and provides an application of tyrosine hydroxylase in a drug for the treatment of collagen-induced arthritis. The expression of TH is down-regulated by using a TH-shRNA plasmid, and the expression of TH is up-regulated by using a plasmid expressing the full-length mouse TH gene. The TH-shRNA plasmid and the plasmid expressing the full-length mouse TH gene are respectively transfected into the CD4 + T cells, observe CD4 + Imbalance of Th17 / Treg in T cells; the results showed that CIA mice CD4 + TH gene silencing in T cells can significantly promote the imbalance of Th17 / Treg, CD4 +Overexpression of TH gene in T cells can significantly inhibit the imbalance of Th17 / Treg (P<0.05).

[0006] Technical solution:

[0007] To achieve the above objectives, this application is implemented through the following technical solutions:

[0008] Application of tyrosine hydroxylase in the treatment of collagen-induced arthritis.

[0009] Furthermore, the collagen-induced arthritis therapeutic drug is an injectable dosage form or a non-injectable dosage form.

[0010] Furthermore, the collagen-induced arthritis therapeutic drug is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

[0011] Furthermore, the collagen-induced arthritis therapeutic drug comprises tyrosine hydroxylase and pharmaceutically acceptable excipients.

[0012] Furthermore, the auxiliary materials include one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

[0013] Principle explanation: Gene interference technology is a technology that shuts down or inhibits the expression of pathogenic genes in a specific way. Its basic principle is to use double-stranded RNA or small molecule RNA as a "guide molecule" to specifically recognize and bind to the mRNA of the target gene, thereby triggering the degradation or translation inhibition process of the mRNA, and ultimately achieving silencing of the gene; gene overexpression is to clone the coding region sequence of the target gene into a plasmid or viral vector, and use the regulatory elements on these vectors to promote the massive transcription and translation of the target gene; TH-shRNA plasmid is used to down-regulate the expression of TH, and a plasmid expressing the full-length mouse TH gene is used to up-regulate the expression of TH. The TH-shRNA plasmid and the plasmid expressing the full-length mouse TH gene are transfected into the CD4 + T cells, CD4 + Expression and secretion changes of Th17 and Treg cell-related nuclear transcription factors and cytokines after TH gene overexpression or silencing in T cells, CD4 + The changes in the number of Th17 and Treg cells after overexpression or silencing of the TH gene in T cells showed that CD4 + TH gene silencing in T cells can significantly promote the imbalance of Th17 / Treg, CD4 +Overexpression of TH gene in T cells can significantly inhibit the imbalance of Th17 / Treg (P<0.05).

[0014] Beneficial effects:

[0015] The present application provides the use of tyrosine hydroxylase in a drug for treating collagen-induced arthritis, which has the following beneficial effects compared with the prior art:

[0016] 1. Overexpression of TH gene in the feet of CIA mice significantly reduced joint swelling, clinical scores, and inflammatory cell infiltration in the joint cavity, while the opposite was true after TH gene silencing;

[0017] 2. After overexpression of TH gene in the feet of CIA mice, the expression and secretion of Th17 cell-related transcription factors and cytokines in the joints and joint tissue supernatants were reduced, and silencing of TH gene further enhanced the function of Th17 cells; after overexpression of TH gene in the feet of CIA mice, the expression and secretion of Treg cell-related cytokines in the joints and joint supernatants were further increased, and silencing of TH gene weakened the function of Treg cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The CD4 + Expression and secretion changes of nuclear transcription factors and cytokines related to Th17 and Treg cells after TH gene overexpression or silencing in T cells; a is a statistical diagram of the relative mRNA expression changes of nuclear transcription factors and cytokines related to Th17 and Treg cells; b is a protein band diagram and expression change statistics of nuclear transcription factors and cytokines related to Th17 and Treg cells; c is a CD4 + Changes in the secretion levels of Th17 and Treg cell-related cytokines in T cell supernatants;

[0019] Figure 2 This application is CIA mouse CD4 + The changes in the number of Th17 and Treg cells after overexpression or silencing of the TH gene in T cells, where a is a flow cytometry graph and b1 is a CD4 + Statistical diagram of the number of Th17 cells after overexpression or silencing of the TH gene in T cells. b2 is the CD4 + Statistical graph of Treg cell number after TH gene overexpression or silencing in T cells. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples, but it should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with the conventional conditions in the art.

[0021] Example 1

[0022] Application of tyrosine hydroxylase in the treatment of collagen-induced arthritis. The collagen-induced arthritis mouse model of this example uses DBA1 / J mice.

[0023] In this example, TH-shRNA plasmid was used to down-regulate the expression of TH, and plasmid expressing the full-length mouse TH gene was used to up-regulate the expression of TH. The TH-shRNA plasmid and the plasmid expressing the full-length mouse TH gene were transfected into the CD4 + T cells, observe CD4 + Imbalance of Th17 / Treg in T cells.

[0024] The specific process is as follows:

[0025] 1. Preparation of collagen-induced arthritis model:

[0026] 1) SPF grade DBA / 1 mice, male, 6-8 weeks old, purchased from the Animal Experiment Center of Nantong University. The experimental animals were kept in a clean animal room with a temperature of 20-24°C, a relative humidity of 50%-60%, and a 12-hour light-dark cycle. They were free to eat and drink, and were used to prepare disease models after three days of adaptation to the environment;

[0027] 2) Weigh 100ug of C II, dissolve it in 50μl 0.1M glacial acetic acid to a concentration of 2mg / ml, and keep it at 4℃ overnight. On day 0, mix 50μl 2mg / ml C II and 50μl CFA in equal volumes, fully emulsify on ice for one hour to make 100μl collagen emulsion. 100μl collagen emulsion was injected at multiple points at the base of the tail of mice in the experimental group, and the blank control group was not treated. On day 21, 50μl C II (2mg / ml) and 50μl IFA were mixed in equal volumes, fully emulsified on ice for one hour to make 100μl collagen emulsion. 100μl collagen emulsion was injected intraperitoneally into mice in the experimental group as stimulating immunity, and the blank control group was not treated. On day 28, 20μl lipopolysaccharide (1mg / ml) was injected intraperitoneally into mice in the experimental group.

[0028] 2. CD4 + T cell magnetic bead sorting and culture:

[0029] 1) Mouse CD4 +T cell sorting kit was purchased from Miltenyi;

[0030] 2) Aseptically take the spleens of mice in the normal control group and the CIA group, place them in a stainless steel mesh, add 4-7 ml of basal culture medium, grind the spleens, and remove fat and connective tissue. Aspirate the cell suspension into a centrifuge tube, add an appropriate amount of basal culture medium, centrifuge at 1800 rpm for 7 minutes, aspirate the middle mononuclear cell layer, add 5-7 ml of red blood cell lysis solution, let stand for 6 minutes, then add basal culture medium to terminate the reaction; centrifuge at 1800 rpm for 7 minutes, add 5-7 ml of basal culture medium to wash the cells, and centrifuge at 1800 rpm for 7 minutes. Wash twice with basal culture medium and count. CD4 + For T cell magnetic bead separation, use a dedicated magnetic bead separation buffer 80μl / 10 7 Resuspend cells and add 10 μl / 10 7 Biotin-Antibody Coktail was mixed and incubated at 2-8°C for 5 min, and then CD4 Microbeads 20 μl / 10 7 Mix well, incubate at 4℃ for 10min, add 1-2ml / 10 7 Wash cells twice with buffer. Use LS column to positively select CD4 + T cells. The cells obtained were 1×10 7 The cells were seeded at a density of 100 / ml in a culture plate, and anti-mouse CD3 and CD28 monoclonal antibodies (1 μg / ml) were added and cultured at 37°C, 5% CO2, and saturated humidity for 48 h to induce CD4 + T lymphocyte proliferation.

[0031] 3. TH overexpression or interference plasmid nuclear transfection CD4 + T cells:

[0032] 1) A plasmid expressing the full length of the mouse TH gene was used as a plasmid for overexpressing the TH gene, and an empty plasmid was used as a control for the TH gene overexpression plasmid. In addition, according to the mouse TH gene sequence and referring to the RNA interference sequence design principle, 4 RNA interference target sequences were designed, DNA oligos containing interference sequences were synthesized, and then annealed to generate double strands, and then directly connected to the lentiviral vector after restriction digestion through the restriction sites contained at both ends, and the ligation products were transferred into the prepared bacterial competent cells. After PCR identification of positive recombinants, sequencing verification was performed, and the sequencing results were compared to confirm that the correct clone was the successfully constructed TH-shRNA plasmid, and then the lentivirus was synthesized. The TH-shRNA interference target sequence is shown in SEQ ID NO.1: 5'-GCACACAGTACATCCGTCA-3', and a plasmid carrying a nonsense sequence is used as an interference control, and the nonsense sequence is shown in SEQ ID NO.2: 5'-TTCTCCGAACGTGTCACGT-3';

[0033] 2) Prepare nuclear transfer solution (2 ml): Mouse T Cell Nucleofector Solution 164.84 μl, 36.36 μl Mouse T Cell Nucleofector Solution supplement, preheat in a 37°C, 5% CO2 incubator for 30 min; preheat AMAXA nuclear transfer complete culture medium; add 1.5 ml complete culture medium to each well of a 12-well plate, preheat in a 37°C, 5% CO2 incubator for 30 min; collect 1×10 lymphocytes after culture 7 / group, 2 groups in total, 500rpm, 5min, centrifugation; collect cell pellets, add 100μl nuclear transfer solution to each of the 2 tubes, and gently suspend the cells; add 4μg plasmid to one tube of cell suspension and gently mix; simulate transfection in the other tube of cell suspension without adding plasmid, the steps are the same. Transfer the cell-plasmid suspension to the AMAXA nuclear transfer cup; debug the nuclear transfer instrument, select X-001 to start the program; put the nuclear transfer cup in the correct position and press the OK button; after the program is completed, immediately add 500μl preheated culture solution to the nuclear transfer cup; immediately move to the preheated 12-well culture plate; culture in a 37℃, 5% CO2 incubator for 48h; after 24-48h, observe the abundance of GFP in the cells under a green fluorescence microscope to evaluate the transfection efficiency. At the same time, the CD4 + RNA and protein were extracted from T cells, and the expression of TH was detected to verify the silencing efficiency.

[0034] 4. Real-time PCR:

[0035] 1) Extract CD4 + T cell total RNA

[0036] Take mouse spleen tissue or isolate and purify CD4 + T cells, add 1ml TRIzol, shake well, let stand at room temperature for 10min, add 200μl chloroform, shake well, let stand at room temperature for 2min, centrifuge at 4℃, 12000rpm for 15min; add 500μl isopropanol, shake well, let stand at room temperature for 10min, centrifuge at 4℃, 12000rpm for 15min, and a white precipitate will be seen at the bottom of the tube; discard the supernatant, add 1ml 75% ethanol (DEPC prepared), and gently wash the precipitate; centrifuge at 4℃7500rpm for 15min; discard the supernatant and dry the precipitate at room temperature; after the precipitate is transparent, dissolve the RNA with 20μl DEPC water, and dissolve it in a 68℃ water bath for 15min;

[0037] 2) RNA content determination

[0038] Take 2 μl RNA sample and add it to 98 μl DEPC water, mix well, use the DEPC water as blank control, and measure OD with nucleic acid protein analyzer. 260 content;

[0039] RNA concentration (μg / ml) = OD 260 Reading × dilution factor × 40 (μg / ml)

[0040] 3) Synthesis of cDNA

[0041] Take 1 μg of total RNA and add DEPC water to 10 μl; add oligo d(T) 18 1μl; gently mix, centrifuge briefly; incubate at 68℃ for 10 minutes, immediately place the centrifuge tube in an ice bath for at least 1 minute, then add the following reagents: 5× buffer 4μl, 10mM dNTP mix 2μl, DEPC water 1.5μl, RNasin 0.5μl; gently mix, centrifuge slightly, and bathe at 42℃ for 5min; then add 1μl of M-MLV reverse transcriptase; gently mix, centrifuge slightly, and bathe at 42℃ for 60min; and bathe at 0℃ for 10min to terminate the reaction. After reverse transcription into cDNA, the changes of each gene were detected by real-time PCR;

[0042] The cDNA samples were diluted 10 times in a row, and the total volume of the PCR reaction was 20 μl, including 2 μl of cDNA template, 0.3 μl of upstream and downstream primers (Table 1), 0.1 μl of Taq enzyme, 10 μl of dNTPmix, 0.4 μl of SYBR Green, and the rest was filled with double distilled water. The PCR reaction conditions were the initial program 95°C, 5 min; 40 cycles: 94°C, 15 s, 62°C, 20 s, 72°C, 20 s. The PCR reaction was performed on an ABI 7500 real-time PCR instrument, using β-actin as an internal reference, and the data were recorded using Rotor-Gene software, version 6.0, using 2 -ΔΔCt The relative expression level of the target gene was calculated using the formula.

[0043] 5.Western blot:

[0044] 1) Sample preparation

[0045] Isolation and purification of CD4 + T cells, add a certain proportion of tissue or cell lysate, shake well, centrifuge the lysate at 12000rpm, 4℃, 15min, boil for 10min, aspirate the supernatant into a new EP tube, and store at -70℃ for later use;

[0046] 2) Protein content determination

[0047] Take 5 μl of the protein standard and the sample to be tested and dilute them with normal saline (the dilution ratio is 1:10), and use a nucleic acid protein analyzer to detect the protein content;

[0048] 3) SDS-polyacrylamide gel electrophoresis

[0049] Use a vertical flat-plate, discontinuous, denaturing polyacrylamide gel electrophoresis system (SDS-PAGE). Prepare 10% separation gel (3.2ml double distilled water, 2.64ml 30% reserve gel, 2ml 1.5mM Tris-HCL, 0.08ml 10% SDS, 0.08ml 10% AP, 4μl TEMED), 4% concentration gel (1.4ml ultrapure water, 0.33ml 30% reserve gel, 2.5ml 1.0mM Tris-HCL, 0.02ml 10% SDS, 0.02ml 10% AP, 2μl TEMED), and add electrophoresis buffer to the upper and lower electrophoresis tanks. Take the supernatant of the tissue lysate from the above different groups and mix with an appropriate amount of buffer, and boil for 3-5min. Take one lane and add pre-stained standard molecular weight protein;

[0050] 4) Western blot detection

[0051] After electrophoresis, constant current wet electrotransfer was performed, and the standard protein lane of the transferred PVDF membrane was cut out. The rest was washed with 1×TBS washing buffer for 5 min × 3 times, and blocked with blocking solution containing 5% skim milk powder at 4°C overnight. Washed with TBS / T washing buffer for 5 min × 3 times, immersed in primary antibodies ROR-γt (1:200), IL-17 (1:200), IL-22 (1:200), Foxp3 (1:200), TGF-β (1:200), IL-10 (1:200) and β-actin (1:5000), and coated at 4°C overnight. Fluorescently labeled goat anti-mouse IgG (1:5000) and fluorescently labeled goat anti-rabbit IgG (1:5000) were incubated at room temperature for 1 hour. Washed with TBS / T washing buffer for 5 min × 3 times, and scanned and analyzed by Odyssey dual infrared laser scanning system.

[0052] 6. Flow cytometry:

[0053] CD4 + T cells were incubated with anti-CD3CD28 antibodies for 48 h, and the recombinant TH gene overexpression or silencing plasmid was transfected into the above cells for 48 h, or CD4 T cells were taken from the spleen of CIA mice injected with TH gene overexpression or silencing lentivirus in the foot. + T cells, 50 ng / ml PMA, 1 mM ionomycin and 2 mM monensin were added to the cells and incubated for 4 h. The cultured CD4 + T cells were placed in EP tubes, centrifuged at 1500rpm for 5min, the supernatant was collected, and resuspended in 100ul PBS, PE-labeled anti-CD25 antibody cells were incubated at 4℃ in the dark for 30min, centrifuged at 1500rpm for 5min, the supernatant was discarded, the cells were washed once with PBS, centrifuged at 1500rpm for 5min, the supernatant was discarded, the cells were fixed and the membrane was broken, centrifuged at 1500rpm for 5min, the supernatant was discarded, the cells were washed once with PBS, centrifuged at 1500rpm for 5min, the supernatant was discarded, 100ul PBS was added to resuspend the cells, APC-labeled anti-Foxp3 antibody and PE-labeled anti-IL-7 antibody were added, incubated at 4℃ in the dark for 30min, centrifuged at 1500rpm for 5min, the supernatant was discarded, the cells were washed once with PBS, centrifuged at 1500rpm for 5min, the supernatant was discarded, the cells were resuspended in PBS, washed twice, 500ul PBS was added, and the cells were detected and analyzed by flow cytometer.

[0054] 7. Statistical analysis:

[0055] All results were expressed as mean ± standard deviation (M ± SD). SPSS10.0 statistical software package was used for data analysis and one-way analysis of variance (ANOVA) was used to test the significance of differences among the groups, with p < 0.05 considered a significant difference.

[0056] The results are as follows Figure 1 As shown, the CD4 + Expression and secretion changes of Th17 and Treg cell-related nuclear transcription factors and cytokines after TH gene overexpression or silencing in T cells; CD4 + T cells were incubated with anti-CD3 and anti-CD28 antibodies for 48 h, and activated CD4 + T cells, total RNA and protein were extracted from cells 48 hours later to observe the CD4 + Changes in the expression of Th17 and Treg cell-related nuclear transcription factors and cytokines after overexpression or silencing of the TH gene in T cells; using real-time PCR,

[0057] Western blot analysis of mouse CD4 + Changes in the mRNA and protein expression of Th17 cell-specific nuclear transcription factor ROR-γt, cytokines IL-17 and IL-22, Treg cell-specific nuclear transcription factor Foxp3, cytokines TGF-β and IL-10 in T cells; ELISA was used to detect CD4 + secretion levels of Th17 and Treg cell-related cytokines in T cell supernatants; Figure 1 Figure a is a statistical chart showing the relative mRNA expression changes of nuclear transcription factors and cytokines related to Th17 and Treg cells. Figure 1 Middle b is the protein band diagram and expression change statistics of nuclear transcription factors and cytokines related to Th17 and Treg cells; Figure 1 c in the middle is CD4 + Changes in the secretion levels of Th17 and Treg cell-related cytokines in T cell supernatants; **p<0.01, compared with the CD4 + T cell comparison; ++ p<0.01, compared with CD4 + T cell comparison; # p<0.05, ## p<0.01, compared with CD4 cells transfected with a plasmid containing nonsense sequence + T cell comparison.

[0058] Figure 2 This application is CIA mouse CD4 + The changes in the number of Th17 and Treg cells after overexpression or silencing of the TH gene in T cells, the CD4 +T cells were incubated with anti-CD3 and anti-CD28 antibodies for 48 h, and activated CD4 + T cells, CD25 was detected by flow cytometry 48 hours later - IL-17 + (Th17) and CD25 + Foxp3 + (Treg) cell number changes; a is the flow cytometry graph, b1 is the CD4 + Statistical diagram of the number of Th17 cells after overexpression or silencing of the TH gene in T cells. b2 is the CD4 + Statistical graph of Treg cell number after TH gene overexpression or silencing in T cells; **p<0.01, compared with CD4 + T cell comparison; ++ p<0.01, compared with CD4 + T cell comparison; ## p<0.01, compared with CD4 cells transfected with a plasmid containing nonsense sequence + T cell comparison.

[0059] CD4 + TH gene silencing in T cells can significantly promote the imbalance of Th17 / Treg, CD4 + Overexpression of TH gene in T cells can significantly inhibit the imbalance of Th17 / Treg (P<0.05). The results of one-way ANOVA showed that the difference between the groups was statistically significant (P<0.05). **p<0.01, compared with the CD4 + T cell comparison; ++ p<0.01, compared with CD4 + T cell comparison; ## p<0.01,

[0060] CD4 cells transfected with a nonsense sequence plasmid + T cell comparison.

[0061] The examples selected in the above materials are for ease of understanding, not to limit the process. People in the same technical field can easily modify the process or transfer it to other cases without creative changes. If these changes also belong to the same claims or similar technologies of the present invention, then the original intention of the invention also includes these changes.

Claims

1. Application of tyrosine hydroxylase in the treatment of collagen-induced arthritis.

2. The use of tyrosine hydroxylase in a drug for treating collagen-induced arthritis according to claim 1, characterized in that: The collagen-induced arthritis therapeutic drug is in the form of an injection or a non-injection dosage form.

3. The use of tyrosine hydroxylase in a drug for treating collagen-induced arthritis according to claim 1, characterized in that: The collagen-induced arthritis therapeutic drug is an injection, tablet, capsule, powder, pill, granule, solution, suspension, syrup, suppository or inhalant.

4. The use of tyrosine hydroxylase in a drug for treating collagen-induced arthritis according to claim 1, characterized in that: The collagen-induced arthritis therapeutic drug comprises tyrosine hydroxylase and pharmaceutically acceptable excipients.

5. The use of tyrosine hydroxylase in a drug for treating collagen-induced arthritis according to claim 4, characterized in that: The auxiliary materials include one or more of diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, and lubricants.

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