Method for promoting synthesis and secretion of growth hormone by bovine gland pituitary cells

Abstract

The invention belongs to the technical field of biological medicines, and relates to a method for promoting synthesis and secretion of growth hormones by bovine gland pituitary cells, in particular to a method for promoting synthesis and secretion of growth hormones by melatonin. In-vitro experiments show that the melatonin can improve the level of adenosine cyclophosphate in cells, promote phosphorylation of PKA protein, promote expression of FOXO1 protein, combine the FOXO1 protein with a promoter of a Gh1 gene and promote synthesis and secretion of growth hormone by combining melatonin receptors in bovine pituitary cells. The application value of the melatonin in the fields of animal growth promotion, growth hormone deficiency treatment and the like is shown.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and includes a method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells. Specifically, it relates to a method for promoting the synthesis and secretion of growth hormone by regulating the melatonin receptor / cAMP / PKA / FOXO1 signaling pathway, and its application in the fields of animal growth promotion, growth hormone deficiency treatment, muscle development regulation, etc. Background Art

[0002] Growth hormone (GH) is a pulsatile hormone synthesized and secreted by pituitary cells. It is encoded by the Gh1 gene, which consists of five exons and four introns and is located on chromosome 17. The primary function of bovine growth hormone protein is to promote the development and growth of bone cells throughout the body. Within the animal body, growth hormone promotes lipid breakdown and protein synthesis in tissues such as the liver, muscle, and adipose tissue, thereby influencing energy metabolism, including the supply and distribution of nutrients and the animal's growth and development. Traditional growth hormone replacement therapy carries the risk of high injection costs and antibody formation.

[0003] The expression of growth hormone is regulated by many factors. Studies have shown that in congenital hypopituitarism, a variety of genes have been identified as pathogenic genes, and FOXO1 is one of the important genes. FOXO1 protein has been confirmed to be involved in the regulation of GH secretion, but its upstream activation mechanism and its association with the melatonin signaling pathway have not yet been clarified. Research on the regulation of pituitary function by melatonin has mostly focused on circadian rhythms, and its molecular pathway for regulating GH secretion through the cAMP / PKA signaling cascade has not been revealed. This patent reveals for the first time the cascade mechanism by which melatonin promotes Gh1 gene transcription through the MTNR1B receptor / cAMP / PKA / FOXO1 signaling axis. The molecular interaction pattern of FOXO1 protein directly binding to the Gh1 gene promoter region (-1526 to -1518bp region) was discovered, and a non-injection GH secretion promotion program based on this pathway was developed. Summary of the Invention

[0004] Technical Solution

[0005] The present invention provides a method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells to solve the above problems.

[0006] A method for promoting the synthesis and secretion of growth hormone in bovine pituitary cells, wherein the method activates the MTNR1A receptor via melatonin, thereby activating the cAMP / PKA / FOXO1 signaling pathway to increase the expression level of the Gh1 gene and the secretion level of growth hormone, wherein the melatonin concentration in the bovine pituitary cells is 100 nM and the treatment time is 6 hours, comprising the following steps:

[0007] SA, optimal concentration and optimal time screening of melatonin

[0008] (1) Isolate and culture primary bovine pituitary cells to a cell density of 3×10 5 / hole;

[0009] (2) Prepare 1 μM melatonin working solution A for cells, 100 nM melatonin working solution B for cells, and 10 nM melatonin working solution C for cells respectively;

[0010] (3) 500 μL of melatonin working solution A, B, or C in step (2) were used to treat the bovine pituitary primary cells cultured in step (1), and the cells were cultured in a CO2 incubator at 37°C. After 2, 4, 6, and 8 hours of drug addition, the cell supernatant samples and adherent cells were collected for subsequent detection;

[0011] (4) extracting RNA from the cell supernatant sample and adherent cells in step (3), and performing qRT-PCR to detect the expression level of GH mRNA in the cells;

[0012] (5) Western blot was used to detect the GH protein secretion level in the cell supernatant samples and adherent cells in step (3);

[0013] (6) collecting the cell culture supernatant in step (3), and using bovine growth hormone and melatonin ELISA kits to determine the secretion levels of growth hormone and melatonin in the culture medium;

[0014] qRT-PCR results showed that 100 nM melatonin treatment for 6 hours in bovine pituitary cells increased intracellular GH mRNA expression by 1.4-fold; WB results showed that 100 nM melatonin treatment for 6 hours significantly increased GH protein secretion by 1.8-fold; ELISA results showed that after 100 nM melatonin administration for 6 hours, the secretion level of growth hormone in the cell supernatant increased significantly by 3-fold compared with the blank control group treated for 6 hours;

[0015] It was shown that the optimal concentration and treatment time of melatonin were 100 nM and 6 h, respectively;

[0016] SB, used siRNA interference and overexpression experiments to verify the role of FOXO1 in melatonin-stimulated growth hormone synthesis and secretion;

[0017] SC, using the JASPAR database (https: / / jaspar.elixir.no), predicted potential transcription factor binding sites in the 2000-bp region upstream of the Gh1 promoter to verify whether FOXO1 regulates Gh1 gene expression by directly binding to the promoter. The results suggested the presence of a FOXO1 binding region, and a dual-luciferase reporter gene system was used to functionally verify the FOXO1 binding region.

[0018] SD verified that melatonin activated the cAMP / PKA signaling pathway to promote FOXO1 expression and regulate the synthesis and secretion of growth hormone. To further verify the role of this pathway, the cells were treated with the cAMP synthesis inhibitor DDA and the PKA activity inhibitor H-89 in combination with melatonin;

[0019] The results showed that melatonin activated the cAMP / PKA signaling pathway in bovine pituitary cells, promoting the expression of FOXO1 protein and thus promoting the synthesis and secretion of growth hormone.

[0020] SE, study the effect of melatonin on the expression of melatonin receptor MTR1A

[0021] This includes immunofluorescence detection of MTR1A expression in bovine pituitary primary cells and blocker combined treatment to verify signal pathway dependency. The blocker combined treatment to verify signal pathway dependency is performed as follows:

[0022] Experimental group 1: 500 μL of 100 nM Luzindole working solution B was used to treat bovine pituitary cells cultured in 24-well plates;

[0023] Experimental group 2: 500 μL of 100 nM 4-P-PDOT working solution B was used to treat bovine pituitary cells cultured in 24-well plates;

[0024] Experimental group 3: 500 μL of complete medium was used to culture the bovine pituitary cells that had been adherently cultured in 24-well plates;

[0025] Control group: 500 μL complete medium, normal culture of bovine pituitary cells adherently cultured in 24-well plates;

[0026] Each group was replicated three times. After culturing in a CO2 incubator at 37°C for 22 hours, the original working solution in each experimental group well was discarded, and 500 μL of 100 nM melatonin working solution B was added to each experimental group well for 6 hours. The control group was treated with 500 μL of complete culture medium for 6 hours. The adherent cells were collected and the expression levels of related genes and proteins were detected by qPCR and Western blot, respectively. The supernatant samples were collected and the cAMP level and growth hormone secretion level were detected using ELISA kits.

[0027] Preferably, the DMEM-F12 complete medium is prepared as follows:

[0028] DMEM-F12 basal medium, penicillin-streptomycin mixture, fetal bovine serum: Add 39.50 mL of DMEM-F12 basal medium, 10.00 mL of 20.0% fetal bovine serum, and 500 μL of penicillin-streptomycin mixture to a 50 mL centrifuge tube, mix well, and store in a refrigerator at 4°C.

[0029] Preferably, the cell-using melatonin working solution is prepared as follows:

[0030] Dissolve 1.16 mg of melatonin in 5 mL of anhydrous ethanol to prepare a 1 mM melatonin working stock solution. Add 1 μL of the melatonin working stock solution to 999 μL of DMEM-F12 basal medium / complete medium to prepare a 1 μM melatonin working solution A. Add 100 μL of melatonin working solution A to 900 μL of DMEM-F12 basal medium / complete medium to prepare a 100 nM melatonin working solution B. Add 100 μL of melatonin working solution B to 900 μL of DMEM-F12 basal medium / complete medium to prepare a 10 nM melatonin working solution C.

[0031] Preferably,

[0032] The primer sequences used in the qRT-PCR are as follows:

[0033]

[0034] Preferably, the bovine pituitary primary cells that have been adherently cultured in step SA (3) are selected for transfection experiments, and the transfection reagent used is a product of Ruibo Company (model: C10511-05); the siRNA negative control (si-NC) and FOXO1 targeted siRNA used for transfection are purchased from Jintuosi (Wuhan); the overexpression control plasmid (OE-NC) and FOXO1 overexpression plasmid are commissioned to Sangon Biotech (Shanghai) to complete the synthesis process.

[0035] Preferably, the primer sequences used in step SB are as follows:

[0036]

[0037]

[0038] Preferably, in the step SD,

[0039] DDA working solution is prepared as follows:

[0040] Dissolve 5 mg of 2',5'-Dideoxyadenosine in 1.063 mL of DMSO to prepare a 20 mM DDA working solution. Add 1 μL of the DDA working solution to 1999 μL of DMEM-F12 basal medium / complete medium to prepare a 10 μM DDA working solution.

[0041] The H-89 working solution is prepared as follows:

[0042] Dissolve 5 mg of H-89 in 560.086 μL of DMSO to prepare a 20 mM H-89 working solution. Add 1 μL of the H-89 working solution to 1999 μL of DMEM-F12 basal medium / complete medium to prepare a 10 μM H-89 working solution.

[0043] Preferably, in step SE,

[0044] The 4-P-PDOT working solution is prepared as follows:

[0045] Dissolve 1.4 mg of 4-P-PDOT in 5 mL of DMSO to prepare a 1 mM 4-P-PDOT working stock solution. Add 1 μL of the 4-P-PDOT working stock solution to 999 μL of DMEM-F12 basal medium / complete medium to prepare a 1 μM 4-P-PDOT working solution A. Add 100 μL of the 4-P-PDOT working solution A to 900 μL of DMEM-F12 basal medium / complete medium to prepare a 100 nM 4-P-PDOT working solution B.

[0046] Luzindole working solution is prepared as follows:

[0047] Dissolve 1.46 mg of Luzindole in 5 mL of DMSO to prepare a 1 mM Luzindole working solution. Add 1 μL of the cell-specific working solution to 999 μL of DMEM-F12 basal medium / complete medium to prepare a 1 μM Luzindole working solution A. Add 100 μL of the cell-specific working solution A to 900 μL of DMEM-F12 basal medium / complete medium to prepare a 100 nM Luzindole working solution B.

[0048] Preferably, the melatonin has a purity of not less than 99.9%.

[0049] Preferably, in step SC, the dual luciferase assay uses a pGL3-Basic reporter plasmid to construct the Gh1 promoter region, which is co-transfected with a FOXO1 overexpression plasmid into 293T cells, and the transcriptional activation ability is evaluated by detecting the firefly luciferase / Renilla luciferase activity ratio.

[0050] The beneficial effects of the present invention are:

[0051] 1. Achieve endogenous regulation of GH and replace the use of exogenous hormones: By activating the melatonin receptor (MTNR1A) and the downstream cAMP / PKA / FOXO1 signaling pathway, it directly promotes the synthesis and secretion of GH in bovine pituitary cells, avoiding the high cost, drug residues, and animal stress reactions caused by the traditional reliance on recombinant GH injections in animal husbandry.

[0052] 2. The molecular mechanism of melatonin regulating GH was revealed for the first time: it was clarified that melatonin activates the cAMP / PKA signaling pathway by binding to the MTNR1A receptor, induces FOXO1 protein expression and directly binds to the Gh1 gene promoter, providing a new theoretical basis for the targeted regulation of GH secretion.

[0053] 3. Significantly improves GH synthesis and secretion efficiency: Experiments showed that 100nM melatonin treatment of bovine pituitary cells for 6 hours increased intracellular GH mRNA expression by 1.4-fold; WB results showed that 100nM melatonin treatment for 6 hours significantly increased GH protein secretion by 1.8-fold; ELISA results showed that after 6 hours of 100nM melatonin administration, the secretion level of growth hormone in the cell supernatant increased significantly by 3-fold compared with the blank control group treated for 6 hours; and knocking down FOXO1 by siRNA completely blocked the secretory effect of MT, confirming the high efficiency and specificity of the regulatory pathway.

[0054] 4. Provide an optimized solution for livestock growth regulation: By adjusting melatonin concentration or using signaling pathway activators (such as cAMP analogs), GH secretion levels can be flexibly controlled, which is suitable for the management of cattle at different growth stages and improves meat production and feed conversion rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The results show that melatonin promotes Gh1 gene expression and growth hormone secretion in bovine pituitary cells.

[0056] Figure 1A shows the expression of Gh1 mRNA in bovine pituitary cells after treatment with 10 nM, 100 nM, and 1 μM melatonin (n=3). B shows the expression of Gh1 mRNA in bovine pituitary cells after 2, 4, 6, and 8 hours of treatment with 100 nM melatonin (n=3). C shows the viability of bovine pituitary cells after treatment with 100 nM melatonin, as assessed by CCK-8 assay (n=3). D shows the expression of Gh1 mRNA in bovine pituitary cells after treatment with 100 nM melatonin (n=3). E shows the expression of GH1 protein in bovine pituitary cells after treatment with 100 nM melatonin (n=3). F shows the secretion of growth hormone in the supernatant of bovine pituitary cells after treatment with 100 nM melatonin (n=3). *P<0.05; **P<0.01; ns: P>0.05.

[0057] Figure 2 The results show that melatonin regulates the synthesis and secretion of GH by regulating FOXO1 expression.

[0058] Figure 2 A shows the expression level of Foxo1 mRNA in bovine pituitary cells after knockdown of FOXO1 (n=3); B shows the expression level of Gh1 mRNA in bovine pituitary cells after knockdown of FOXO1 (n=3); C shows the expression levels of FOXO1 protein and GH1 protein in bovine pituitary cells after knockdown of FOXO1; D shows the secretion level of growth hormone in the supernatant of bovine pituitary cells after knockdown of FOXO1; E shows the expression level of Foxo1 mRNA in bovine pituitary cells after overexpression of FOXO1 (n=3); F shows the expression level of Gh1 mRNA in bovine pituitary cells after overexpression of FOXO1 (n=3); G shows the expression level of FOXO1 protein and GH1 protein in bovine pituitary cells after overexpression of FOXO1; H shows the secretion level of growth hormone in the supernatant of bovine pituitary cells after overexpression of FOXO1; I shows the expression level of Foxo1 in bovine pituitary cells after administration of 100 nM MT. mRNA expression (n=3); J is the expression level of FOXO1 protein in bovine pituitary cells after administration of 100 nM MT; I is the analysis of the binding between FOXO1 and the Gh1 promoter by dual-luciferase reporter gene assay (n=3); J is the predicted binding site between FOXO1 and the Gh1 gene promoter (Sorce>10). *P<0.05; **P<0.01; ns: P>0.05.

[0059] Figure 3 The results show that melatonin regulates the synthesis and secretion of GH through the cAMP / PKA / FOXO1 signaling pathway.

[0060] Figure 3A shows the cAMP level in bovine pituitary cells after 100 nM MT administration (n=6); B shows the expression of PKA and p-PKA proteins in bovine pituitary cells after 100 nM MT administration (n=3); C shows the expression of Foxo1 mRNA in bovine pituitary cells after co-treatment with MT and DDA (n=3); D shows the protein expression of p-PKA, PKA, and FOXO1 in bovine pituitary cells after co-treatment with MT and DDA (n=3); E shows the expression of Gh1 mRNA in bovine pituitary cells after co-treatment with MT and DDA (n=3); F shows the secretion level of GH in bovine pituitary cells after co-treatment with MT and DDA (n=3);

[0061] G represents the expression of Foxo1 mRNA in bovine pituitary cells after co-treatment with MT and H-89 (n=3); H represents the expression of FOXO1 protein in bovine pituitary cells after co-treatment with MT and H-89 (n=3); I represents the expression of Gh1 mRNA in bovine pituitary cells after co-treatment with MT and H-89 (n=3); J represents the secretion level of growth hormone in bovine pituitary cells after co-treatment with MT and H-89 (n=3). *P<0.05, **P<0.01, ***P<0.001; ns: P>0.05.

[0062] Figure 4 The results show that melatonin regulates the synthesis and secretion of GH through the MTNR1A receptor.

[0063] Figure 4 A shows the expression of MTNR1A protein in bovine pituitary cells after administration of 100 nM MT (n=3); B shows the spatial expression of MTNR1A in bovine pituitary cells detected by immunofluorescence after administration of 100 nM MT; C shows the expression of Gh1 mRNA in bovine pituitary cells after co-treatment of MT and 4-P-PDOT (n=3);

[0064] D is the cAMP concentration in bovine pituitary cells after co-treatment with MT and 4-P-PDOT (n=6); E is the expression levels of p-PKA, PKA and FOXO1 proteins in bovine pituitary cells after co-treatment with MT and 4-P-PDOT (n=3); F is the expression level of Foxo1 mRNA in bovine pituitary cells after co-treatment with MT and 4-P-PDOT (n=3); G is the secretion level of growth hormone in the supernatant of bovine pituitary cells after co-treatment with MT and 4-P-PDOT (n=3); *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0065] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0066] Experimental reagents

[0067] Anhydrous ethanol, xylene and physiological saline (Boyu Pharmaceutical), reverse transcription and quantification reagents (iScience), total RNA extraction reagent (YaZyme Biotechnology), protein-free rapid blocking buffer, 12.5% PAGE gel kit and protein loading buffer and protein marker (YaZyme Biotechnology), GH and FOXO1 antibodies (Sanying Biotechnology), MTNR1B antibody (Abclone), DMEM-F12 basal medium (Vivacell), fetal bovine serum (Lonsera), CCK-8 kit, trypsin for cells (Biyuntian), double antibody (Gibco), bovine GH and MT ELISA kits (Shanghai ELISA Biotechnology Co., Ltd.), cAMP detection kit (Wuhan Elaruite Biotechnology Co., Ltd.), DMSO (Solerbo), melatonin, 4-4P-PDOT, Luzindole, 2',5'-Dideoxyadenosine and H-89 (MCE), PKA and p-PKA antibodies (Affinity), FOXO1 siRNA (Wuhan Jintuosi), FOXO1 overexpression plasmid (Sangon Biotech), transfection kit (Guangzhou Ruibo), cell-based experimental consumables (Nice Biotech)

[0068] Experimental equipment

[0069] Refrigerator (Haier), surgical instruments (Kangli Medical), electric grinding pestle (TGrinder), centrifuge (Thermo), fluorescence quantitative PCR instrument (Mona), microplate reader (Thermo Fisher), PCR instrument (Kunpeng Gene), ultra-micro nucleic acid analyzer (Pruibo), fluorescence microscope (Zeiss), incubator (Thermo), chemiluminescence analyzer (Tianneng Life Science), electronic balance (Lesder), electrophoresis instrument (Bio-Rad).

[0070] Example 1 Experimental pretreatment

[0071] Fresh pituitary tissue of Simmental cattle was obtained from slaughterhouses around Changchun City, Jilin Province. Primary cells were isolated by referring to the method of Wu Hao et al., Study on the Effect of MicroRNA-181b on FSH Secretion of Bovine Pituitary Cells (Jilin Animal Husbandry and Veterinary Medicine, 2017, 38(06):5-7). The obtained cell suspension was inoculated into a six-well plate, and 1 mL of cell suspension and 1 mL of DMEM-F12 complete medium were added to each well. The cells were cultured in a 37°C, 5% CO2 incubator for 4 days to promote cell adhesion and growth. On the 4th day, all cells were digested and resuspended, and then evenly distributed into a pre-prepared 24-well plate. 500 μL of complete medium was added to each well. The cells were cultured under the same conditions for 24 hours until the cell concentration reached about 3×10 5 After that, the subsequent experimental treatment was carried out.

[0072] In the above embodiment, DMEM-F12 complete medium was prepared as follows:

[0073] DMEM-F12 basal culture medium and penicillin-streptomycin mixture (double antibody) were purchased from Vivacell, and fetal bovine serum was purchased from Lonsera.

[0074] To a 50 mL centrifuge tube, add 39.50 mL of DMEM-F12 basal medium, 10.00 mL of fetal bovine serum (20.0%), and 500 μL of penicillin-streptomycin mixture (double antibody), mix well, and store in a refrigerator at 4°C.

[0075] Preparation of working fluid

[0076] Melatonin, 4-P-PDOT, DDA, and H-89 were purchased from MCE.

[0077] Prepare melatonin working solution for cells: Dissolve 1.16 mg of melatonin in 5 mL of anhydrous ethanol to prepare a 1 mM melatonin working stock solution. Add 1 μL of the melatonin working stock solution to 999 μL of DMEM-F12 basal medium / complete medium to prepare a 1 μM melatonin working solution A. Add 100 μL of melatonin working solution A to 900 μL of DMEM-F12 basal medium / complete medium to prepare a 100 nM melatonin working solution B. Add 100 μL of melatonin working solution B to 900 μL of DMEM-F12 basal medium / complete medium to prepare a 10 nM melatonin working solution C.

[0078] Prepare DDA working solution: Dissolve 5 mg of 2',5'-Dideoxyadenosine in 1.063 mL of DMSO to prepare a 20 mM DDA working solution. Add 1 μL of the DDA working solution to 1999 μL of DMEM-F12 basal medium / complete medium to prepare a 10 μM DDA working solution.

[0079] Prepare H-89 working solution: Dissolve 5 mg of H-89 in 560.086 μL of DMSO to prepare a 20 mM H-89 working solution. Add 1 μL of the H-89 working solution to 1999 μL of DMEM-F12 basal medium / complete medium to prepare a 10 μM H-89 working solution.

[0080] Prepare 4-P-PDOT working solution: Dissolve 1.4 mg of 4-P-PDOT in 5 mL of DMSO to prepare a 1 mM 4-P-PDOT working stock solution. Add 1 μL of the 4-P-PDOT working stock solution to 999 μL of DMEM-F12 basal medium / complete medium to prepare a 1 μM 4-P-PDOT working solution A. Add 100 μL of the 4-P-PDOT working solution A to 900 μL of DMEM-F12 basal medium / complete medium to prepare a 100 nM 4-P-PDOT working solution B.

[0081] Prepare Luzindole working solution: Dissolve 1.46 mg of Luzindole in 5 mL of DMSO to prepare a 1 mM Luzindole working solution. Add 1 μL of the working solution to 999 μL of DMEM-F12 basal medium / complete medium to prepare a 1 μM Luzindole working solution A. Add 100 μL of working solution A to 900 μL of DMEM-F12 basal medium / complete medium to prepare a 100 nM Luzindole working solution B.

[0082] Example 2 Study on the promotion of growth hormone synthesis and secretion in bovine pituitary cells by melatonin

[0083] To clarify the regulatory effect of melatonin on the synthesis and secretion of growth hormone in the pituitary gland, this study used melatonin to treat bovine pituitary cells and screened for the optimal concentration and time. The specific operation is as follows:

[0084] 2.1 Screening of appropriate melatonin treatment concentration

[0085] Experimental design:

[0086] Experimental group 1: 500 μL of melatonin working solution A was used to treat bovine pituitary cells cultured in 24-well plates;

[0087] Experimental group 2: 500 μL of melatonin working solution B was used to treat bovine pituitary cells cultured in 24-well plates;

[0088] Experimental group 3: 500 μL of melatonin working solution C was used to treat bovine pituitary cells cultured in 24-well plates;

[0089] Control group: 500 μL of DMEM-F12 complete medium was used to treat the bovine pituitary cells that had been cultured adherently in a 24-well plate;

[0090] Each group was replicated for 3 wells and cultured in a 37°C CO2 incubator. After 2, 4, 6, and 8 hours of drug addition, the cell supernatant samples and adherent cells were collected for subsequent testing to screen the optimal treatment concentration and treatment time.

[0091] 2.2 RNA extraction

[0092] (1) Add 1 mL of TRlzol to the cell supernatant sample and adherent cells collected from each well of the 24-well plate in step 2.1. After standing at room temperature for two minutes, transfer them to 1.5 mL centrifuge tubes. Add 200 μL of chloroform to each tube, shake vigorously up and down with both hands for 15 seconds, stand at room temperature for 2-3 minutes, and centrifuge at 12000 g for 15 minutes.

[0093] (2) After centrifugation, the liquid in the tube automatically separates into three layers: the upper layer is a colorless aqueous phase, the middle layer is a thin white protein layer, and the lower layer is a colored organic phase. 400 μL to 500 μL of the upper transparent liquid from the two centrifuge tubes are respectively drawn into two new 1.5 mL centrifuge tubes;

[0094] (3) Add equal volumes of isopropanol to two centrifuge tubes containing the supernatant, mix thoroughly by pipetting, place in a 4°C refrigerator to precipitate for 10-30 min, and centrifuge at 12,000 g for 10 min.

[0095] (4) Remove the supernatant and wash the pellet with 1 mL of 75% ice-cold ethanol, vortex for 30 seconds, and centrifuge at 7500 g for 5 minutes.

[0096] (5) Remove the supernatant, open the tube cap in a clean bench, let the precipitate stand and dry for 3-5 minutes, and then aspirate the supernatant with a pipette tip;

[0097] (6) Add 20 μL of 0.1% DEPC water to each tube to dissolve the RNA precipitate and aliquot.

[0098] 2.3 qRT-PCR detection of gene expression

[0099] (1) Use the FastKing cDNA kit for reverse transcription. First, add 2 μL of 5×gDNA Buffer and 8 μL of the total RNA extracted in step 3.1 to each PCR tube. After pipetting evenly, incubate at 42°C for 3 minutes to obtain working solution A.

[0100] (2) Add 2 μL of 10× King RT Buffer, 1 μL of FastKing RT Enzyme Mix, 2 μL of FQ-RT Primer Mix, and 5 μL of RNase-Free ddH2O to each PCR tube to prepare working solution B;

[0101] (3) Mix the working solutions a and b thoroughly, incubate at 42°C for 15 min and then at 95°C for 3 min to obtain cDNA;

[0102] (4) 2× SuperReaL PreMix PLus 10 μL, Forward Primer 0.5 μL, Reverse Primer 0.5 μL, cDNA 1 μL, ddH2O 8 μL. qRT-PCR was performed according to this system to detect the mRNA expression level of the Gh1 gene in primary bovine pituitary cells, with GAPDH as the internal reference gene.

[0103] Experimental data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 8 software. Differences between groups were assessed using a two-tailed t-test or one-way analysis of variance (ANOVA). The significance level was set at *P < 0.05, **P < 0.01, and ***P < 0.001.

[0104]

[0105]

[0106] When screening treatment concentrations, the expression of Gh1mRNA in bovine pituitary cells treated with 100nM melatonin was significantly increased ( Figure 1 A). When screening the treatment time, the blank control group was used as the benchmark. After 6 hours of treatment with 100nM melatonin, the expression of Gh1 mRNA increased significantly ( Figure 1 B). Repeated results showed that after 6 hours of 100nM melatonin administration, the expression of Gh1 mRNA in cells was significantly increased compared with the blank control group 6 hours later ( Figure 1 .D).

[0107] The experimental results showed that 100nM melatonin treatment for 6 hours in bovine pituitary cells could increase the expression of GH mRNA in the cells by 1.4 times, proving that the optimal treatment concentration and treatment time of melatonin are 100nM and 6 hours, respectively.

[0108] 2.4 CCK-8 assay for cell proliferation

[0109] Experimental design: The cell pretreatment procedure in Example 1 was repeated. After the cell concentration in the 24-well plate reached about 3×10 5 / well, the bovine pituitary cells were treated with 100 nM melatonin for 6 hours.

[0110] For experimental group A1, bovine pituitary cells treated with 100 nM melatonin for 6 hours were digested and resuspended. 2000 cells were plated per well of a 96-well plate. 180 μL of melatonin working solution B and 20 μL of CCK-8 solution were added to each well.

[0111] The blank group A2 was tested by adding 100 nM melatonin and CCK-8 solution for cells in the same amount as the experimental group, but without adding cells.

[0112] For control group B1, bovine pituitary cells treated with 100 nM melatonin for 6 hours were digested and resuspended. 2,000 cells were plated per well of a 96-well plate. 180 μL of complete culture medium and 20 μL of CCK-8 solution were added to each well.

[0113] The blank control group B2 was added with the corresponding amount of complete culture medium and CCK-8 solution as the control group, but no cells were added.

[0114] Each group was repeated three times. After incubation in the cell culture incubator for another hour, the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific). A standard curve was established and calibrated using the OD values of (A1-A2) and (B1-B2). The calculated value was used to reflect the cell proliferation level.

[0115] CCK-8 results showed that 100nM melatonin had no effect on cell viability ( Figure 1 Therefore, this study selected 100 nM for 6 h for subsequent experiments.

[0116] 2.5Western blot

[0117] (1) After completing the cell culture in step 2.1, discard the supernatant and wash with 1× PBS. Add 120 μL of protein lysis buffer and 1.2 μL of protease inhibitor to each well to fully lyse the cells.

[0118] (2) After lysis, centrifuge at 16,000 g for 10 minutes, aspirate the supernatant and transfer it to a new centrifuge tube for protein concentration determination. Then, add protein loading buffer, mix well, and denature at 95°C for 10 minutes.

[0119] (3) Prepare gel using a PAGE gel rapid preparation kit.

[0120] (4) After the PAGE gel is assembled, add 10 μL of protein sample.

[0121] (5) Set up the electrophoresis apparatus at 90V constant voltage for 30 min until the protein sample enters the lower gel, then run at 120V constant voltage until the sample band reaches the bottom of the gel, and end the electrophoresis.

[0122] (6) Take out the gel and place the gel and PVDF membrane in the red and black clamps in order. Set up the electrophoresis apparatus and run it at a constant current of 220 mA to transfer the protein to the PVDF membrane.

[0123] (7) Block with protein-free rapid blocking solution for 15 minutes.

[0124] (8) Incubate with primary antibody at room temperature for 2 h.

[0125] (9) After incubation, the primary antibody was recovered and washed three times with 1× TBST, each time for 10 min.

[0126] (10) Place the PVDF membrane in the diluted secondary antibody and incubate at room temperature for 1 h.

[0127] (11) After incubation, wash the membrane three times with 1× TBST, each time for 10 min.

[0128] (12) Prepare the luminescent working solution in a 1:1 ratio according to the instructions.

[0129] (13) Use a chemiluminescence imager to capture protein bands.

[0130] (14) The obtained protein bands were analyzed using Image J.

[0131] Protein lysis buffer, protease inhibitors, PAGE gel rapid preparation kit gel, protein-free rapid blocking buffer, 1×TBST and 1×PBS were purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.

[0132] Primary antibodies used were rabbit polyclonal antibodies against β-tubulin (purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.) and polyclonal antibodies against GH (purchased from Wuhan Sanying Biotechnology Co., Ltd.). Secondary antibodies used were HRP-conjugated goat anti-rabbit IgG (purchased from Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.). β-tubulin was used as an internal reference protein to calculate the relative expression of GH protein in each group.

[0133] WB results showed that 100nM melatonin treatment for 6 hours significantly increased the GH protein secretion level by 1.8 times ( Figure 1 .E).

[0134] 2.6ELISA detection of growth hormone and melatonin concentrations

[0135] Collect the cell culture supernatant in step 2.1 and use bovine growth hormone and melatonin ELISA kits to measure the secretion levels of growth hormone and melatonin in the culture medium.

[0136] The bovine growth hormone ELISA kit model used in the enzyme-linked immunosorbent assay is mlbio ml105904, and the bovine melatonin ELISA kit model is mlbio ml106525.

[0137] ELISA results showed that after 6 hours of administration of 100nM melatonin, the secretion level of growth hormone in the cell supernatant increased significantly by 3 times compared with the blank control group treated for 6 hours ( Figure 1 .F).

[0138] Example 3 Melatonin promotes the expression of FOXO1 in bovine pituitary cells

[0139] To verify the key role of FOXO1 in melatonin-induced growth hormone synthesis and secretion, siRNA interference and overexpression experiments were conducted. Adherently cultured bovine pituitary primary cells were used for transfection experiments.

[0140] For detailed transfection experiments, please refer to [1] Wang Wenhua. Study on the regulation of bovine follicle-stimulating hormone secretion by GnRH analogs through miR-488[D]. Jilin University, 2020. DOI: 10.27162 / d.cnki.gjlin.2020.000937.

[0141] The transfection reagent used was a product from Ribo (model: C10511-05). The siRNA negative control (si-NC) and FOXO1-targeting siRNA used for transfection were purchased from Genetron (Wuhan). The overexpression control plasmid (OE-NC) and FOXO1 overexpression plasmid were synthesized by Sangon Biotechnology (Shanghai).

[0142] Bovine pituitary cells were transfected with si-NC, FOXO1 siRNA, OE-NC and OE-FOXO1 plasmids, respectively. The mRNA and protein expression levels of Gh1 and FOXO1 were detected by RT-qPCR and Western blot, and the secretion of growth hormone in the culture medium was also detected.

[0143] The specific steps of RT-qPCR and Western blot are shown in Example 2.

[0144] The primary anti-FOXO1 polyclonal antibody in Western blot detection was purchased from Wuhan Tri-Ting Biotechnology Co., Ltd. β-Tubulin was used as the internal reference protein to calculate the relative expression levels of GH protein and FOXO1 protein in each group.

[0145] The primers used in RT-qPCR are:

[0146]

[0147] Experimental Subject Description:

[0148] The cells used in overexpression and interference experiments were primary bovine pituitary adherent cells, and FOXO1 gene expression was interfered with by transfection of plasmids or siRNA.

[0149] The following table shows the recommended experimental group settings:

[0150]

[0151] The results show:

[0152] Transfection with FOXO1 siRNA significantly downregulated the mRNA and protein levels of FOXO1 and Gh1, and the secretion of growth hormone was significantly decreased ( Figure 2 AD).

[0153] After transfection of FOXO1 overexpression plasmid, the mRNA and protein levels of FOXO1 and Gh1 increased significantly, and the secretion level of growth hormone was significantly improved ( Figure 2 EH).

[0154] In addition, further detection found that after 6 hours of treatment with 100nM melatonin, the mRNA expression of FOXO1 in cells was significantly upregulated compared with the control group (6 hours). Figure 2 I), and its protein expression was also significantly enhanced ( Figure 2 J), suggesting that melatonin can upregulate FOXO1 expression, thereby regulating the synthesis and secretion of growth hormone.

[0155] To further verify whether FOXO1 regulates Gh1 gene expression by directly binding to its promoter, the JASPAR database (https: / / jaspar.elixir.no) was used to predict potential transcription factor binding sites in the 2000 bp region upstream of the Gh1 promoter. The results suggested that there was a FOXO1 binding region ( Figure 2 L).

[0156] To verify whether the binding site plays a regulatory role, a dual-luciferase reporter gene system was used for functional verification. The experiment was performed by Ruibo Biotechnology (Guangzhou). A pGL3-Basic reporter vector containing the Gh1 promoter sequence was constructed and co-transfected with a FOXO1 overexpression vector into 293T cells.

[0157] Experimental setup:

[0158] Reporter fluorescent gene: firefly luciferase;

[0159] Internal reference fluorescent gene: Renilla luciferase;

[0160] Detection ratio: The luciferase activity ratio (FLuc / RLuc) reflects the degree of transcriptional activation.

[0161] Plasmid construction and processing were commissioned by Sangon (Shanghai, China). The specific steps were as follows: First, the bovine Gh1 gene promoter sequence was ligated into pGL3-Basic to construct a reporter gene vector. The reporter gene vector and the bovine FOXO1 overexpression vector were then co-transfected into 293T tool cells. The firefly luciferase gene was used as the reporter fluorescent gene for this vector, and the Renilla luciferase gene was used for fluorescence activity calibration. Finally, the fluorescence activities of the two were divided, and the relative value was used to analyze the interaction between FOXO1 and the Gh1 gene promoter.

[0162] The results showed that FOXO1 overexpression could significantly enhance the luciferase activity of Gh1 promoter ( Figure 2 K), indicating that FOXO1 can directly bind to the Gh1 promoter and enhance its transcriptional activity, confirming that it is the core downstream regulatory factor for melatonin-mediated increase in Gh1 gene expression.

[0163] In summary, FOXO1 plays a key transcriptional regulatory role in the process of melatonin promoting Gh1 expression, and it can be used as a potential target for regulating growth hormone levels in animals.

[0164] Example 4: Melatonin activates the cAMP / PKA signaling pathway to promote FOXO1 expression and regulate growth hormone synthesis and secretion

[0165] To explore the role of cAMP / PKA signaling pathway in melatonin-induced growth hormone synthesis and secretion, 100 nM melatonin was first added to bovine pituitary cells for 6 hours, and the changes in intracellular cAMP levels and PKA signaling pathway were detected.

[0166] 4.1 cAMP level detection

[0167] Experimental design: The cell pretreatment procedure in Example 1 was repeated. After the cell concentration in the 24-well plate reached about 3×10 5 / well, the bovine pituitary cells were treated with 100 nM melatonin for 6 hours.

[0168] After removing the culture medium from bovine pituitary cells treated with 100 nM melatonin for 6 hours, 0.1 M HCl lysis buffer was added to the cells. After incubation at room temperature for 10 minutes, the cells were centrifuged at 1000 g for 10 minutes, and the supernatant was collected. Intracellular cAMP content was detected using a cAMP ELISA kit (E-EL-0056, Wuhan Elaruite), and readings were completed using a Swiss Sunrise F50 fully automatic microplate reader at a wavelength of 450 nm. Differential expression analysis was performed using the culture medium from bovine pituitary cells treated with a control group for 6 hours as a control.

[0169] The experimental results showed that melatonin treatment could significantly increase the intracellular cAMP level ( Figure 3 .A), suggesting that melatonin can activate cAMP signaling.

[0170] 4.2 Detection of PKA and p-PKA Expression

[0171] Western blot analysis was used to measure the expression of PKA and its phosphorylated form, p-PKA, in bovine pituitary cells treated with 100 nM melatonin for 6 hours as described in 4.1. Primary antibodies against PKA and p-PKA provided by Affinity Biosciences were used. Images were taken using a chemiluminescent imager (Tianneng Life Sciences), and grayscale values of protein bands were analyzed using ImageJ software.

[0172] The results showed that melatonin treatment significantly increased the p-PKA / PKA ratio, suggesting that the PKA pathway was activated ( Figure 3 .B).

[0173] 4.3 Blocker combination treatment to verify signaling pathway dependence

[0174] To further verify the role of this pathway, this study used the cAMP synthesis inhibitor DDA and the PKA activity inhibitor H-89 combined with melatonin to treat cells as follows:

[0175] Experimental design: Repeat the cell pretreatment procedures in Example 1. After the cell concentration in the 24-well plate reaches about 3×10 5 / well, perform subsequent experiments.

[0176] Experimental group 1: 500 μL of 10 μM DDA working solution was used to treat bovine pituitary cells cultured in 24-well plates;

[0177] Experimental group 2: 500 μL of 10 μM H-89 working solution was used to treat bovine pituitary cells cultured in 24-well plates;

[0178] Control group: 500 μL complete culture medium (solvent), normal culture of bovine pituitary cells adherently cultured in 24-well plates.

[0179] Each group was incubated in triplicate at 37°C in a CO2 incubator for 22 hours. The original working solution in each experimental group was discarded, and 500 μL of 100 nM melatonin working solution was added to each experimental group for 6 hours. Adherent cells were harvested and expression levels of related genes and proteins were analyzed by qPCR and Western blot, respectively. Supernatant samples were collected and growth hormone secretion levels were analyzed using an ELISA kit.

[0180] DDA+melatonin treatment group: significantly reduced the expression of FOXO1 and Gh1 mRNA induced by melatonin ( Figure 3 .C, E), while reducing growth hormone secretion ( Figure 3 .F), and inhibited the upregulation of p-PKA and FOXO1 protein expression ( Figure 3 .D).

[0181] H-89+melatonin treatment group: significantly inhibited the upregulation of FOXO1 and Gh1 mRNA induced by melatonin ( Figure 3 .G, I), inhibiting the secretion of growth hormone ( Figure 3 .H) and reduced FOXO1 protein expression ( Figure 3 .J).

[0182] The experimental results show that melatonin promotes the expression of FOXO1 protein by activating the cAMP / PKA signaling pathway in bovine pituitary cells, thereby promoting the synthesis and secretion of growth hormone.

[0183] Example 5 Melatonin promotes the synthesis and secretion of growth hormone through the MTR / cAMP / PKA / FOXO1 signaling pathway

[0184] To clarify the molecular mechanism by which melatonin regulates growth hormone synthesis through its membrane receptor, this study focused on the key role of MTR1A in the melatonin-induced cAMP / PKA pathway.

[0185] 5.1 Effect of melatonin on the expression of melatonin receptor MTR1A

[0186] Repeat the cell pretreatment procedure in Example 1 until the cell concentration in the 24-well plate reaches about 3×10 5 After 4 h, the bovine pituitary cells were treated with 100 nM melatonin for 6 h, and the expression and localization of MTR1A protein were detected by Western blot.

[0187] The specific steps of Western blot are shown in Example 2.

[0188] Immunofluorescence was used to detect the expression of MTR1A in primary bovine pituitary cells. The steps are as follows:

[0189] (1) Washing: Wash twice by centrifugation with 1xPBS (1000 rpm, 5 min), and prepare cell smears directly.

[0190] (2) Fixation: Cover the cells with 4% neutral formaldehyde fixative and place at 4°C for 15 minutes.

[0191] (3) Blocking. Block the cells with blocking solution for 30 minutes.

[0192] (5) Primary antibody binding. Incubate at room temperature for 1 hour. Rinse three times with 1xPBST, 5 minutes each rinse.

[0193] (6) Secondary antibody binding. For immunofluorescence, incubate with secondary antibody at room temperature in the dark for 1 h. Rinse with 1xPBST three times, 5 min each time, and then rinse once with distilled water.

[0194] (7) Nuclear staining: Add DAPI dye and incubate at room temperature in the dark for 5 minutes. Rinse with 1xPBST three times, 5 minutes each time, and then rinse once with distilled water.

[0195] (8) Sealing and testing: Add one drop of sealing agent, seal the slide, and examine under a fluorescence microscope.

[0196] The primary MTR1A polyclonal antibody for Western blot and immunofluorescence assays was purchased from Affinity. β-Tubulin was used as an internal reference protein to calculate the relative expression of MTR1A protein in each group. Immunofluorescence staining kits with anti-rabbit Cy3 and DAPI were purchased from Beyotime.

[0197] The results showed that melatonin treatment could activate MTR1A receptor ( Figure 4 .A). Immunofluorescence results showed that MTR1A receptors were expressed on the cell membrane surface ( Figure 4 .B).

[0198] 5.2 Blocker combination treatment to verify signaling pathway dependence

[0199] To verify that melatonin activates downstream pathways through the melatonin receptor MTR1A. Experimental design:

[0200] Experimental group 1: 500 μL of 100 nM Luzindole working solution B was used to treat bovine pituitary cells cultured in 24-well plates;

[0201] Experimental group 2: 500 μL of 100 nM 4-P-PDOT working solution B was used to treat bovine pituitary cells cultured in 24-well plates;

[0202] Experimental group 3: 500 μL of complete medium (solvent) was used to culture the bovine pituitary cells adherently cultured in a 24-well plate.

[0203] Control group: 500 μL complete culture medium (solvent), normal culture of bovine pituitary cells adherently cultured in 24-well plates.

[0204] Each group was incubated in a 37°C CO2 incubator for 22 hours with three replicates. The original working solution in each experimental group was discarded, and 500 μL of 100 nM melatonin working solution B was added to each experimental group for 6 hours. The control group was treated with 500 μL of complete culture medium for 6 hours. Adherent cells were harvested and the expression levels of relevant genes and proteins were analyzed by qPCR and Western blot, respectively. Supernatant samples were collected and analyzed for cAMP levels and growth hormone secretion using ELISA kits.

[0205] The primer sequences for qPCR are disclosed here.

[0206] 4-P-PDOT+melatonin treatment group: The results showed that MTR1B specific receptor inhibition by 4-P-PDOT had no significant effect on Gh1 mRNA expression ( Figure 4 .C).

[0207] Luzindole+melatonin treatment group: The results showed that MTR1A specific receptor inhibition by Luzindole could significantly inhibit Gh1 mRNA expression activity ( Figure 4 .C), significantly reduced the promoting effect of MT on FOXO1 mRNA expression ( Figure 4 .F), and also significantly reduced the promoting effect of MT on the expression of p-PKA and FOXO1 proteins ( Figure 4 .E); inhibiting MTR activity and significantly reducing the increase in cAMP and growth hormone secretion in cells caused by MT ( Figure 4 .DG).

[0208] The experimental results show that melatonin activates the cAMP / PKA / FOXO1 pathway by binding to the MTR1A receptor on the surface of the bovine pituitary cell membrane, rather than the MTR1B receptor, thereby promoting the synthesis and secretion of growth hormone.

[0209] In summary, melatonin activates the intracellular cAMP signaling pathway by binding to the melatonin receptor MTR1A, inducing PKA protein phosphorylation and increasing FOXO1 expression. FOXO1 protein directly binds to the Gh1 gene promoter, promoting Gh1 mRNA expression and enhancing growth hormone synthesis and secretion.

[0210] 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 embodied 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 illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0211] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells, characterized in that: The method activates the MTNR1A receptor through melatonin, thereby activating the cAMP / PKA / FOXO1 signaling pathway to increase the Gh1 gene expression level and the growth hormone secretion level, wherein the concentration of melatonin used to treat bovine pituitary cells is 100 nM and the treatment time is 6 hours, comprising the following steps: SA, optimal concentration and optimal time screening of melatonin (1) Isolate and culture primary bovine pituitary cells to a cell density of 3×10 5 / hole; (2) Prepare 1 μM melatonin working solution A for cells, 100 nM melatonin working solution B for cells, and 10 nM melatonin working solution C for cells respectively; (3) 500 μL of melatonin working solution A, B, or C in step (2) were used to treat the bovine pituitary primary cells cultured in step (1), and the cells were cultured in a CO2 incubator at 37°C. After 2, 4, 6, and 8 hours of drug addition, the cell supernatant samples and adherent cells were collected for subsequent detection; (4) extracting RNA from the cell supernatant sample and adherent cells in step (3), and performing qRT-PCR to detect the expression level of GH mRNA in the cells; (5) Western blot was used to detect the GH protein secretion level in the cell supernatant samples and adherent cells in step (3); (6) collecting the cell culture supernatant in step (3), and using bovine growth hormone and melatonin ELISA kits to determine the secretion levels of growth hormone and melatonin in the culture medium; qRT-PCR results showed that 100 nM melatonin treatment for 6 hours in bovine pituitary cells increased intracellular GH mRNA expression by 1.4-fold; WB results showed that 100 nM melatonin treatment for 6 hours significantly increased GH protein secretion by 1.8-fold; ELISA results showed that after 100 nM melatonin administration for 6 hours, the secretion level of growth hormone in the cell supernatant increased significantly by 3-fold compared with the blank control group treated for 6 hours; It was shown that the optimal concentration and treatment time of melatonin were 100 nM and 6 h, respectively; SB, used siRNA interference and overexpression experiments to verify the role of FOXO1 in melatonin-stimulated growth hormone synthesis and secretion; SC, using the JASPAR database (https: / / jaspar.elixir.no), predicted potential transcription factor binding sites in the 2000-bp region upstream of the Gh1 promoter to verify whether FOXO1 regulates Gh1 gene expression by directly binding to the promoter. The results suggested the presence of a FOXO1 binding region, and a dual-luciferase reporter gene system was used to functionally verify the FOXO1 binding region. SD verified that melatonin activated the cAMP / PKA signaling pathway to promote FOXO1 expression and regulate the synthesis and secretion of growth hormone. To further verify the role of this pathway, the cells were treated with the cAMP synthesis inhibitor DDA and the PKA activity inhibitor H-89 in combination with melatonin; The results showed that melatonin activated the cAMP / PKA signaling pathway in bovine pituitary cells, promoting the expression of FOXO1 protein and thus promoting the synthesis and secretion of growth hormone. SE, study the effect of melatonin on the expression of melatonin receptor MTR1A This includes immunofluorescence detection of MTR1A expression in bovine pituitary primary cells and blocker combined treatment to verify signal pathway dependency. The blocker combined treatment to verify signal pathway dependency is performed as follows: Experimental group 1: 500 μL of 100 nM Luzindole working solution B was used to treat bovine pituitary cells cultured in 24-well plates; Experimental group 2: 500 μL of 100 nM 4-P-PDOT working solution B was used to treat bovine pituitary cells that had been cultured adherently in a 24-well plate; Experimental group 3: 500 μL of complete medium was used to culture the bovine pituitary cells that had been adherently cultured in 24-well plates; Control group: 500 μL complete medium, normal culture of bovine pituitary cells adherently cultured in 24-well plates; Each group was replicated three times. After culturing in a CO2 incubator at 37°C for 22 hours, the original working solution in each experimental group well was discarded, and 500 μL of 100 nM melatonin working solution B was added to each experimental group well for 6 hours. The control group was treated with 500 μL of complete culture medium for 6 hours. The adherent cells were collected and the expression levels of related genes and proteins were detected by qPCR and Western blot, respectively. The supernatant samples were collected and the cAMP level and growth hormone secretion level were detected using ELISA kits.

2. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: The DMEM-F12 complete culture medium was prepared as follows: DMEM-F12 basal culture medium, penicillin-streptomycin mixture, fetal bovine serum: Add 39.50 mL of DMEM-F12 basal culture medium, 10.00 mL of 20.0% fetal bovine serum, and 500 μL of penicillin-streptomycin mixture to a 50 mL centrifuge tube, mix well, and store in a 4°C refrigerator.

3. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: The cell melatonin working solution was prepared as follows: Dissolve 1.16 mg of melatonin in 5 mL of anhydrous ethanol to prepare a 1 mM melatonin working solution for cells. Add 1 μL of melatonin working solution for cells to 999 μL of DMEM-F12 basal medium / complete medium to prepare melatonin working solution A for cells with a concentration of 1 μM; add 100 μL of melatonin working solution A for cells to 900 μL of DMEM-F12 basal medium / complete medium to prepare melatonin working solution B for cells with a concentration of 100 nM; add 100 μL of melatonin working solution B for cells to 900 μL of DMEM-F12 basal medium / complete medium to prepare melatonin working solution C for cells with a concentration of 10 nM.

4. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: The primer sequences used in the qRT-PCR are as follows:

5. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: The bovine pituitary primary cells that had been cultured adherently in step SA (3) were selected for transfection experiments. The transfection reagent used was a product of Ribo. The siRNA negative control si-NC and FOXO1 targeted siRNA used for transfection were purchased from Genetron. The overexpression control plasmid OE-NC and FOXO1 overexpression plasmid were synthesized by Sangon Biotechnology.

6. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: The primer sequences used in step SB are as follows:

7. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: In the step SD, DDA working solution is prepared as follows: Dissolve 5 mg of 2',5'-Dideoxyadenosine in 1.063 mL of DMSO to prepare a 20 mM DDA working solution. Add 1 μL of DDA working stock solution to 1999 μL of DMEM-F12 basal medium / complete medium to prepare a DDA working solution with a concentration of 10 μM; The H-89 working fluid is prepared as follows: 5 mg of H-89 was dissolved in 560.086 μL of DMSO to prepare a 20 mM H-89 working stock solution. Add 1 μL of H-89 working stock solution to 1999 μL of DMEM-F12 basal medium / complete medium to prepare a 10 μM H-89 working solution.

8. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: In the step SE, The 4-P-PDOT working solution is prepared as follows: Dissolve 1.4 mg of 4-P-PDOT in 5 mL of DMSO to prepare a 1 mM 4-P-PDOT working solution for cells. Add 1 μL of 4-P-PDOT working solution to 999 μL of DMEM-F12 basal medium / complete medium to prepare 1 μM 4-P-PDOT working solution A. Add 100 μL of 4-P-PDOT working solution A to 900 μL of DMEM-F12 basal medium / complete medium to prepare 100 nM 4-P-PDOT working solution B. Luzindole working solution is prepared as follows: Dissolve 1.46 mg of Luzindole in 5 mL of DMSO to prepare a Luzindole working solution with a concentration of 1 mM. Add 1 μL of cell working stock solution to 999 μL of DMEM-F12 basal medium / complete medium to prepare Luzindole working solution A with a concentration of 1 μM. Add 100 μL of cell working solution A to 900 μL of DMEM-F12 basal medium / complete medium to prepare Luzindole working solution B with a concentration of 100 nM.

9. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: The melatonin purity is not less than 99.9%.

10. The method for promoting the synthesis and secretion of growth hormone by bovine pituitary cells according to claim 1, characterized in that: In step SC, the dual luciferase assay uses a pGL3-Basic reporter plasmid to construct the Gh1 promoter region, which is co-transfected with a FOXO1 overexpression plasmid into 293T cells. The transcriptional activation ability is assessed by detecting the firefly luciferase / Renilla luciferase activity ratio.

Images