Use of antibacterial peptide PG-1 in promoting hormone secretion of ovarian granulosa cells

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of antibacterial peptide PG-1 in promoting hormone secretion of ovary granulosa cells. In order to explore the influence of PG-1 on hormone secretion of ovary granulosa cells and the mechanism thereof, and clarify the physiological regulation of PG-1 on female reproduction, the application takes pig ovary granulosa cells as a cell model, and finds that PG-1 can up-regulate mRNA expression levels of estrogen E2 secretion related genes (CYP11A1, HSD3B1 and HSD17B1), and then promote estradiol (E2) secretion of large follicle granulosa cells, which indicates that the PG-1 has a promoting effect on hormone secretion of ovary granulosa cells, and has important potential application value in female reproduction. The application provides a strong theoretical basis and practical basis for further improving the reproductive function of female animals and promoting the research and development of corresponding drugs, and has important research and development value and development significance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of antimicrobial peptide PG-1 in promoting hormone secretion by ovarian granulosa cells. Background Technology

[0002] Animal reproductive performance is influenced by a variety of factors. Besides the nutrition provided by feed, the reproductive capacity of mammals is primarily determined by the fertility of the female animal itself. The quality of ovarian follicle development significantly impacts reproductive functions such as estrus, ovulation, fertilization, and pregnancy. Follicle maturation mainly depends on the maturation of the oocyte and the proliferation and differentiation of granulosa cells. Granulosa cells play a crucial supporting and protective role in oocyte maturation, and the estrogen and progesterone secreted by granulosa cells are essential for maintaining follicle development. Recent studies have found that hormones secreted by granulosa cells not only regulate animal reproduction but also influence the body's immune and inflammatory responses. Therefore, understanding the secretion regulation and metabolic regulation of hormones from granulosa cells is of great significance.

[0003] Estrogen secretion plays a crucial role in reproductive function and development. The antimicrobial peptide PG-1 possesses antimicrobial and immunomodulatory activities, but its effects on female reproduction are poorly studied, and there are currently no reports on how PG-1 influences hormone secretion by ovarian granulosa cells. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention uses porcine ovarian granulosa cells as a cell model to explore the effects of PG-1 on hormone secretion in in vitro cultured porcine ovarian granulosa cells and its mechanism, and to elucidate the physiological regulatory role of PG-1 in female reproduction.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides the use of the antimicrobial peptide PG-1 in the preparation of a drug that promotes the secretion of estrogen by ovarian granulosa cells.

[0006] Preferably, the estrogen is estradiol (E2).

[0007] Preferably, the ovarian granulosa cells are large follicle granulosa cells.

[0008] Preferably, the antimicrobial peptide PG-1 promotes estrogen secretion by ovarian granulosa cells by upregulating the mRNA expression level of estrogen secretion-related genes.

[0009] More preferably, the estrogen secretion-related genes include CYP11A1, HSD3B1, and HSD17B1.

[0010] This invention uses primary cultured porcine ovarian granulosa cells as the research object to investigate the effect of the antimicrobial peptide PG1 on the E2 secretion level of porcine ovarian granulosa cells, in order to reveal its physiological function in sow reproductive development. After isolating and culturing porcine large follicle granulosa cells, different concentrations (0, 10 µg / mL) of PG1 were added to the in vitro culture medium for 6 h. E2 secretion levels were detected using ELISA, and the mRNA expression levels of key factors in E2 synthesis, such as aromatase and hydroxysteroid dehydrogenase, were detected using qPCR. The results showed that 10 µg / mL of PG1 significantly increased the secretion levels of E2 and P4 from porcine ovarian granulosa cells, and significantly upregulated the mRNA expression levels of CYP11A1, HSD3B1, and HSD17B1. These results indicate that a PG1 concentration of 10 µg / mL is beneficial for E2 secretion from porcine ovarian granulosa cells, suggesting that the antimicrobial peptide PG1 has a promoting effect on hormone secretion from ovarian granulosa cells and has important potential application value in female reproduction.

[0011] A second aspect of the present invention provides a drug for promoting estrogen secretion by ovarian granulosa cells, the drug having an antimicrobial peptide PG-1 as the main active ingredient.

[0012] Preferably, the drug further includes pharmaceutically acceptable excipients. These excipients may be diluents, binders, lubricants, disintegrants, solubilizers, stabilizers, and some pharmaceutical matrices that are applicable in the pharmaceutical field; they may also be functional pharmaceutical excipients available in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some novel pharmaceutical polymers, such as cyclodextrin, chitosan, polylactic acid (PLA), polyglycolic acid-polylactic acid copolymer (PLGA), hyaluronic acid, etc.

[0013] Preferably, the dosage form of the drug includes tablets, granules, capsules, pellets, sustained-release formulations, oral liquid preparations, powders for injection, and injections. The dosage form refers to commonly used clinical dosage forms, and the drug preparation can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally), allowing for flexible administration via various routes such as oral, subcutaneous, intramuscular, and intravenous injection. If certain drugs are unstable under gastric conditions, they can be formulated as enteric-coated tablets.

[0014] Compared with the prior art, the beneficial effects of the present invention are: Ovarian development and reproductive performance in female animals are closely related. Follicle development and oocyte quality are closely related to the growth status and hormone secretion function of granulosa cells. Estradiol (E2) secreted by granulosa cells can regulate oocyte growth and follicle development. This invention has found that PG-1 can upregulate the mRNA expression levels of E2 secretion-related genes (CYP11A1, HSD3B1, and HSD17B1), thereby promoting estradiol (E2) secretion by granulosa cells in large follicles. This indicates that PG-1 has a promoting effect on hormone secretion by ovarian granulosa cells and has important potential application value in female reproduction. This invention provides a strong theoretical and practical foundation for further improving the reproductive function of female animals and promoting the development of corresponding drugs, and has significant research value and development significance. Attached Figure Description

[0015] Figure 1 This represents the amount of estradiol secreted by granulosa cells after PG-1 treatment. Figure 2 The mRNA expression level of estrogen E2 secretion-related genes in granulosa cells after PG-1 treatment. Detailed Implementation

[0016] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0017] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0018] Study on the effect of PG-1 on hormone secretion from cultured porcine ovarian granulosa cells (1) Isolation and culture of primary porcine follicular granulosa cells Fresh pig ovaries were collected from a local slaughterhouse in Foshan and placed in a thermos containing PBS solution at 28-35℃. They were transported back to the laboratory within 1.5 hours and washed three times with sterile PBS solution to remove surface blood. Follicular fluid was then extracted from large follicles (3-6 mm in diameter) using an 18G needle. After allowing the follicular fluid to settle for 15 minutes, the supernatant of follicular fluid and red blood cells was discarded. Granulosa cells were washed twice with F-12 cell culture medium containing 1% penicillin (100 U / mL) and streptomycin (0.1 mg / mL). Cell clumps were then dispersed evenly using a pipette. The cells were centrifuged at 500 rpm for 5 minutes, the supernatant was discarded, and the cells were mixed with 1 mL of F12 culture medium. 20 µL of the cell solution was diluted 10-fold and thoroughly mixed. Another 10 µL of the cell solution was stained with phenol blue and counted using a TC20 automated cell counter (BioRad). Cells were diluted to 1×10⁶ cells using F12 culture medium containing 1% penicillin (100 U / mL) and streptomycin (0.1 mg / mL) and 10% calf serum. 6 Cells / mL were collected, and then 500µL of cell solution was evenly spread into each of the 24-well adherent cell culture plates. Finally, the cells were placed in a CO2 incubator and incubated at 37°C and 5% CO2 for 24 hours.

[0019] (2) Effect of PG-1 on estradiol (E2) secretion in large follicle granulosa cells: porcine primary granulosa cells (cell concentration 5×10⁻⁶) 4 After treatment with 10 μg / mL PG-1 (Shanghai Toppeptide Biotechnology Co., Ltd.) for 6 h, the supernatant was collected, and estradiol secretion was detected using an ELISA kit (Enzyme-Linked Biotechnology). Independent samples t-tests were used to analyze the statistical data, which are expressed as mean ± standard deviation (p < 0.05, n ≥ 3, different letters indicate statistical significance).

[0020] (3) Effects of PG-1 on the mRNA expression levels of estrogen E2 secretion-related genes (CYP11A1, HSD3B1 and HSD17B1): Granulocytes (GCs) treated with PG-1 were collected by centrifugation. Total RNA was extracted from GCs using the TaKaRa MiniBEST Universal RNA Extraction Kit (No. 9767, TaKaRa, Kusatsu, Shiga, Japan) according to the manufacturer's instructions, and RNA was quantified using a NanoPhotometer NP80 Touch (IMPLEN, Germany). For each sample, cDNA was prepared by reverse transcription using 500 ng of total RNA. Specifically, reverse transcription was performed using the PrimeScript™ RT Kit (No. RR047A, TaKaRa) with a gDNA eraser (Perfect Real-Time). The reverse transcription process was performed using the Biometra T Professional Standard 96 Gradient Thermycler (Biometra, Germany). Quantitative PCR™ II (Tli RNaseH Plus) (TaKaRa) was then performed using TBGreen® Premix Ex Taq. The reaction contained 200 nM of each primer (see Table 1) and 10% cDNA. The entire reaction was performed for 40 cycles on a Light Cycler 480 II real-time PCR system (Roche, Basel, Switzerland). The quantitative PCR employed a two-step amplification protocol with a denaturation temperature of 95 °C and an annealing temperature of 60 °C. Melting curve analysis after RT-qPCR confirmed the specificity of the PCR amplification products by the presence of a single peak. The size of the PCR products was confirmed by gel electrophoresis, and no visible primer dimers were observed.

[0021] Throughout the experiment, primers were designed according to the standard of crossing introns or targeting exon-exon connections to ensure that the cDNA template was not contaminated with gDNA. At the same time, the primer efficiency and PCR amplification efficiency were determined by serially diluting the cDNA template in the range of 5% to 105%. In addition, the GAPDH gene (housekeeping gene) was selected as an internal control to standardize the expression data, and the relative expression level of the gene was calculated using the 2-ΔΔCt method.

[0022] Table 1 Primer sequences for genes related to upregulating estrogen E2 secretion (CYP11A1, HSD3B1, and HSD17B1) like Figure 1 , 2As shown, 10 µg / mL PG1 significantly increased the secretion of E2 and P4 by porcine ovarian granulosa cells and significantly upregulated the mRNA expression levels of CYP11A1, HSD3B1, and HSD17B1. These results indicate that a PG1 concentration of 10 µg / mL is beneficial for E2 secretion by porcine ovarian granulosa cells, and has significant potential application in regulating the physiological functions of female reproduction.

[0023] In summary, PG-1 can upregulate the mRNA expression levels of estrogen E2 secretion-related genes (CYP11A1, HSD3B1 and HSD17B1), thereby promoting estradiol (E2) secretion in large follicle granulosa cells, and has important potential application value in female reproduction.

[0024] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. The application of antimicrobial peptide PG-1 in the preparation of drugs that promote estrogen secretion from ovarian granulosa cells, characterized in that, The estrogen is estradiol, and the antimicrobial peptide PG-1 promotes estrogen secretion by ovarian granulosa cells by upregulating the mRNA expression level of estrogen secretion-related genes; the ovarian granulosa cells are large follicular granulosa cells.

2. The application according to claim 1, characterized in that, The estrogen secretion-related genes include CYP11A1, HSD3B1, and HSD17B1.

3. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The dosage forms of the drug include tablets, granules, capsules, pills, oral liquid preparations, and injections.

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