Applications of GALANIN family members in shortening labor or promoting parturition in mice

Abstract

This invention discloses the application of GALANIN family members in the preparation of products for shortening labor and promoting parturition in mice. The GALANIN family members are GALP peptides or SPEXIN peptides. The amino acid sequence of the GALP peptide is APAHRGRGGWTLNSAGYLLGPVLPVSSKADQGRKRDSALEILDLWKIIDGLPYSHSPRMT, and the amino acid sequence of the SPEXIN peptide is NWTPQAMLYLKGAQ-NH2. Injecting GALP peptides or SPEXIN peptides into mice awaiting parturition significantly shortens the labor process, reducing it from 203.22 min to 107.30 min, greatly improving parturition efficiency. It is anticipated that the application of GALP peptides or SPEXIN peptides to other mammals can also improve their parturition efficiency, providing new solutions and ideas for mammalian parturition problems.

Description

Technical Field

[0001] This invention relates to the field of animal reproduction and breeding, and in particular to the application of GALANIN family members in shortening the labor process or promoting delivery in mice. Background Technology

[0002] Among the currently known mechanisms of labor regulation, the labor process can be divided into three stages: the first stage, the initiation of labor; the second stage, the maintenance of labor; and the third stage, the termination of labor. In the first stage, labor begins with uterine contractions and ends with complete cervical dilation and effacement. Progesterone and estrogen play the main regulatory roles, characterized by a decrease in progesterone levels (which maintain uterine stillness during pregnancy) and a rise in estrogen levels, relieving the inhibition of uterine contractions and initiating labor. The second stage begins with complete cervical dilation and ends with fetal delivery. In this stage, prostaglandins and oxytocin, among other factors, jointly regulate rhythmic uterine contractions, facilitating successful fetal delivery. The third stage begins after fetal delivery and ends with placental expulsion. Oxytocin plays a major role in this stage. However, the exact mechanisms governing labor regulation are still under investigation and require further research.

[0003] GALANIN is a neuropeptide containing 29 amino acids (30 amino acids in humans), widely expressed in the central and peripheral nervous systems to regulate various biological functions, including pain perception, learning, memory, and appetite. GALANIN selectively binds to different GAL receptors (GALR) in various tissues to regulate their function; GALR consists of GALR1, GALR2, and GALR3. Members of the GALANIN family include GALANIN, SPEXIN (and SPEXIN2 in non-mammalian vertebrates), KISSPEPTIN, GALP, ALARIN, and GAMP, among which GALANIN, SPEXIN, and GALP bind to GALR to exert their effects. GALP is a non-C-terminal modified peptide composed of 60 amino acids. Its residues (9-21) are identical to those of GALANIN (1-13), while residues (1-24) and (41-53) are highly conserved across species. SPEXIN is a neuropeptide composed of 14 amino acids. The mature sequences of SPEXIN and GALANIN are highly similar across different vertebrate species and exhibit conserved structures in many species. However, the role of GALANIN family members in regulating mammalian parturition has not been reported. Summary of the Invention

[0004] The purpose of this invention is to provide applications of GALANIN family members in shortening labor or promoting delivery in mice, in order to address the problem of long labor and dystocia during delivery, and to provide new ideas and methods for solving the problem of delivery in mammals.

[0005] According to a first aspect of the invention, the use of GALANIN family members in the preparation of products for shortening the labor process in mice is provided, wherein the GALANIN family member is a GALP peptide or a SPEXIN peptide, wherein the amino acid sequence of the GALP peptide is APAHRGRGGWTLNSAGYLLGPVLPVSSKADQGRKRDSALEILDLWKIIDGLPYSHSPRMT, and the amino acid sequence of the SPEXIN peptide is NWTPQAMLYLKGAQ-NH2. This application can significantly shorten the labor process in mice, thereby improving labor efficiency and providing a new solution and method for addressing parturition problems in mammals.

[0006] According to a second aspect of the invention, the use of GALANIN family members in the preparation of products for promoting parturition in mice is provided, wherein the GALANIN family member is a GALP peptide or a SPEXIN peptide, wherein the amino acid sequence of the GALP peptide is APAHRGRGGWTLNSAGYLLGPVLPVSSKADQGRKRDSALEILDLWKIIDGLPYSHSPRMT, and the amino acid sequence of the SPEXIN peptide is NWTPQAMLYLKGAQ-NH2. Thus, this application can promote parturition in animals, and in particular can significantly shorten the parturition process, thereby improving parturition efficiency.

[0007] The beneficial effects of this invention are as follows: By injecting GALP peptide or SPEXIN peptide into mice awaiting delivery, the delivery process can be significantly shortened, from 203.22 min to 107.30 min, greatly improving delivery efficiency. It is anticipated that the application of GALP peptide or SPEXIN peptide to other mammals can also improve their delivery efficiency, providing new solutions and ideas for mammalian delivery problems. Detailed Implementation

[0008] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Unless otherwise specified, the chemical substances, proteins, enzymes, or reagent kits used in the present invention are all commercially available.

[0009] Example 1: Exploring the drug concentration by administering the drug via gavage.

[0010] (1) Animal preparation:

[0011] Breeding female mice are caged together in the afternoon and checked for culls the next morning. If a cull is found, the day after 12 noon is counted as 0.5 days. They usually give birth 18.5 days after mating (days post-coitum, dpc). At 13.5 dpc, each female mouse is placed in a separate cage for acclimatization and transition.

[0012] (2) Drug preparation:

[0013] a. Control group: 10% DMSO, 40% PEG300, 5% Tween-80, 45% Saline.

[0014] b. Receptor inhibitor treatment group: GALANIN receptor inhibitors (M40 and HT2157, both at the same concentration) were added to the control group as the solvent, with concentrations of 10... -4 mM, 10 -3 mM, 10 -2 mM, 10 -1 mM, 4×10 -1 mM.

[0015] M40 is an effective non-selective GALANIN receptor inhibitor that competitively inhibits GALR1 and GALR2. It is a GALR1 and GALR2 inhibitor. This M40 inhibitor was purchased from R&D Systems, catalog number: 3425.

[0016] HT2157, also known as SNAP 37889, is a selective, high-affinity competitive GALR3 antagonist. It is a GALR3 inhibitor. This HT2157 inhibitor was purchased from MedChemExpress, catalog number: HY-100717.

[0017] (3) Gavage treatment:

[0018] After observing the mother mouse give birth to the first pup, immediately administer 100 μL of the drug via gavage. Then, after 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, and 50 minutes, administer the drug for the second, third, fourth, fifth, sixth, seventh, and eighth times, for a total of eight gavages, each time administering 100 μL of the drug.

[0019] Record the medication administered via gavage, the mother mouse's number, and the time of each gavage, and label the cage with the mother mouse's number. Immediately after gavage, move away from the cage and avoid loud talking. During the mother mouse's labor, record the birth time of each pups. 55 minutes after the 8th gavage (300 minutes after the 1st gavage), record the number of live and dead pups born in each cage. Then immediately euthanize the mother mouse and check if there are any unborn pups in her uterus; record the number of unborn pups for each mother mouse.

[0020] The results are shown in Table 1. The results indicate that the completion rate of delivery in the receptor inhibitor treatment group was lower than that in the control group, 10 -1 mM and 4×10 -1 The total delivery time, average time required for each pup to be born, and number of remaining fetuses in the uterus were significantly higher in the mM concentration group than in the control group. P <0.05), the number of live offspring in the cage was significantly lower than that in the control group ( P <0.05), there was no significant difference in the number of dead pups in the cages ( P >0.05). This indicates that 10 -1 mM and 4×10 -1 mM represents the optimal treatment concentration. Combined use of GALANIN receptor inhibitors (GALR1, GALR2, GALR3) (M40+HT2157) during parturition in pregnant mice resulted in dystocia and prolonged parturition. This indicates that GALANIN can significantly affect parturition in mice, such as influencing the duration of labor and the number of live pups.

[0021] Table 1. Effects of combined gavage administration of three GALANIN receptor inhibitors on parturition in pregnant mice.

[0022]

[0023] Note: Data represents the mean ± standard error; different lowercase letters in the same column indicate significant differences. P <0.05), the same lowercase letters in the same column indicate no significant difference (P>0.05).

[0024] Example 2: Slow-release drug delivery via implanted osmotic pump.

[0025] (1) Animal preparation:

[0026] The temperature for raising mice is controlled at 20-26℃ using an air conditioning system. Relative humidity is maintained at 50%-60% using a humidifier. Feed and water are added weekly at fixed times, and at other times based on the amount of feed remaining in the hopper, 3-4 times per week. Breeding females are provided with sufficient breeding feed, and 0.5-1 gram of sunflower seeds are fed daily at 9:00 AM to supplement vitamin E and energy. Breeding females are placed in the cage in the afternoon, and the cull is checked the following morning. If a cull is found, the day after 12:00 PM is counted as 0.5 days. Giving birth generally occurs 18.5 days after mating (days post-coital, dpc).

[0027] (2) Drug preparation:

[0028] The optimal treatment concentration determined in Example 1 is 10. -1 mM and 4×10 -1 mM, evaluated from a safety and cost perspective, at 10 -1 The drug concentration in the osmotic pump was calculated based on mM to ensure that there was a sufficient drug dose when the osmotic pump permeated the animal's body. This confirmed that the drug concentration in the osmotic pump was 41 mM.

[0029] a. Control group (NC): 100% DMSO.

[0030] b. Receptor inhibitor treatment group (RI): M40 (GALR1 and GALR2 inhibitors) and HT2157 (GALR3 inhibitor) were dissolved in DMSO to a final concentration of 41 mM.

[0031] c. GALANIN peptide treatment group: GALANIN was dissolved in DMSO to a final concentration of 41 mM.

[0032] d. SPEXIN peptide treatment group: SPEXIN was dissolved in DMSO to a final concentration of 41 mM.

[0033] e. GALP peptide treatment group: GALP was dissolved in DMSO to a final concentration of 41mM.

[0034] The amino acid sequence of the GALANIN peptide is (SEQ ID No:1): GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2, where -NH2 indicates c-terminal amidation modification; the GALANIN peptide was purchased from Abcam, catalog number ab141153.

[0035] The amino acid sequence of the SPEXIN peptide is (SEQ ID No:2): NWTPQAMLYLKGAQ-NH2, where -NH2 indicates c-terminal amidation modification; the SPEXIN peptide was purchased from MedChemExpress, catalog number HY-P1723.

[0036] The amino acid sequence of the GALP peptide is (SEQ ID No:3): APAHRGRGGWTLNSAGYLLGPVLPVSSKADQGRKRDSALEILDLWKIIDGLPYSHSPRMT; the GALP peptide was synthesized by GenScript.

[0037] (3) Injection osmosis pump:

[0038] Using Alzet osmotic pumps (Alzet Osmotic Pumps, 1003D), each pump is infused with approximately 100 μL of drug, released slowly at a rate of 1 μL per hour. The effective duration of action for the osmotic pump is 72 hours. First, weigh the empty pump and pump cap. Using a 1 ml syringe with the blunt-tipped needle attached to the capsule pump, draw up the drug solution, expelling any air from the needle. Hold the capsule osmotic pump upright, insert the blunt-tipped needle to the bottom of the pump, and slowly push the drug solution into the pump until a small amount overflows from the pump outlet. Carefully remove the needle, fully insert the pump cap into the pump, wipe away any excess solution, and finally weigh the entire pump to calculate the infused solution volume. The calculated infused liquid volume should exceed 90% of the pump capacity specified in the instructions. If this is not achieved, it indicates excessive air in the pump, requiring refilling.

[0039] (4) Implantation of an osmotic pump:

[0040] The osmotic pump was infused at room temperature, then immersed in 0.9% saline solution for 4-6 hours before starting. At 13.5 dpc, each female mouse was placed in a separate cage for acclimatization. At 17.5 dpc, the animals were anesthetized, and a 0.5cm transverse incision was made subcutaneously on the animal's back. Tissue forceps were used to create a capsule-sized space from the subcutaneous tissue at the incision site into the animal's back. The capsule containing the drug solution was surgically implanted, and the wound was sutured. After implantation, the female mice were returned to their original transparent cages, and the parturition process was recorded using a camera. The onset and end times of parturition were closely monitored, and the total number of mice born and the number of live mice in the cage were recorded after each female mouse gave birth. At 20.5 dpc, the osmotic pump stopped releasing the drug, and the female mice were immediately euthanized. The uterus was examined to determine if there were any unborn pups remaining, indicating dystocia.

[0041] (5) Experimental results:

[0042] By infusing Alzet osmotic pumps with three GALANIN receptor inhibitors, M40 and HT2157, respectively, and then implanting the drug-loaded pumps into the backs of pregnant mice, the experimental results of the combined sustained-release of the three GALANIN receptor inhibitors (M40+HT2157) on the parturition of pregnant mice are shown in Table 2. The results show that combined treatment of pregnant mice with the three GALANIN receptor inhibitors significantly reduced the proportion of mice that completed parturition. 60% of the mice did not give birth after an average onset time of 18.5 days (dpc) and did not begin parturition before the end of the effective period of the drugs (20.5 dpc), and there were still fetuses in the uterus. 40% of the mice that gave birth had significantly higher onset time of parturition, total parturition duration, and time required for the birth of each pup than the control group mice. P <0.05). This indicates that the combined use of GALANIN receptor inhibitors (GALR1, GALR2, GALR3) (M40+HT2157) during parturition in pregnant mice leads to dystocia in most female mice. Although some female mice are still able to give birth, the time to onset of parturition and the duration of labor are prolonged.

[0043] Table 2. Effects of combined sustained-release GALANIN receptor inhibitors on parturition in pregnant mice.

[0044]

[0045] Note: Data are expressed as mean ± standard error; different lowercase letters in the same row indicate significant differences. P <0.05), identical lowercase letters in the same row indicate no significant difference ( P >0.05).

[0046] Since the results in Table 2 indicate that the inhibitors of the three GALANIN receptors (GALR1, GALR2, and GALR3) (M40+HT2157) can lead to prolonged labor and even dystocia in female mice, we selected GALANIN, SPEXIN, and GALP peptides that can bind to GALR1, GALR2, and GALR3 receptors and administered them in pregnant mice via sustained-release administration. We hypothesized that these peptides might shorten the labor process or promote delivery in mice and further verify this hypothesis.

[0047] Therefore, by infusing 100 μL of GALANIN, SPEXIN, and GALP peptides into an Alzet osmotic pump, and then implanting the drug-loaded osmotic pump into the back of pregnant mice, the experimental results of the sustained release of the three GALANIN family members (GALANIN, SPEXIN, and GALP peptides) on the parturition of pregnant mice are shown in Table 3: Comparison of total parturition duration: In the GALANIN treatment group, compared with the control group (NC), the total parturition duration decreased from 203.22 min in the control group to 90.20 min, and the difference was significant. P<0.05); In the SPEXIN treatment group, compared with the control group (NC), the total labor duration decreased from 203.22 min in the control group to 125.10 min, and the difference was statistically significant. P <0.05); In the GALP treatment group, compared with the control group (NC), the total labor duration decreased from 203.22 min in the control group to 107.30 min, and the difference was statistically significant. P <0.05). Comparison of time required for each pup to be born: In the GALANIN treatment group, compared with the control group (NC), the time required for each pup to be born decreased from 13.52 min in the control group to 5.61 min, and the difference was statistically significant. P <0.05); In the SPEXIN treatment group, compared with the control group (NC), the time required for each pup to be born decreased from 13.52 min in the control group to 8.25 min, and the difference was statistically significant. P <0.05); In the GALP treatment group, compared with the control group (NC), the time required for each pup to be born decreased from 13.52 min in the control group to 8.44 min, and the difference was statistically significant. P <0.05). However, there were no significant differences between the GALANIN, SPEXIN, and GALP treatment groups and the control group (NC) in terms of the completion rate of delivery, time of onset of labor, total number of pups, and number of live pups. P >0.05).

[0048] The results showed that treating female mice with the three GALANIN family members separately significantly reduced the total birthing time and the time required for each pup to be born. P The levels were <0.05), and there were no significant differences in the time to onset of parturition, total number of offspring, and number of live offspring. This indicates that the use of three members of the GALANIN family (GALANIN, SPEXIN, and GALP) during parturition in pregnant mice can significantly shorten the labor process without affecting the time to onset of parturition, total number of offspring, and number of live offspring.

[0049] Table 3. Effects of sustained-release GALANIN family members on parturition in pregnant mice.

[0050]

[0051] Note: Data represents the mean ± standard error; different lowercase letters in the same column indicate significant differences. P <0.05), identical lowercase letters in the same column indicate no significant difference ( P >0.05).

[0052] In conclusion, injecting GALANIN family members (especially GALANIN, SPEXIN, and GALP) into mice awaiting delivery can effectively shorten the labor process without affecting the onset of delivery, total number of pups, or live pups. This demonstrates that GALANIN family members can effectively shorten the labor process and promote delivery in mice, and this method can be applied to other mammals such as pigs, cattle, and sheep to effectively shorten the labor process and improve delivery efficiency.

Claims

1. Use of a GALANIN family member for the preparation of a product for shortening the delivery in mice, wherein, The GALANIN family member is a GALP polypeptide having an amino acid sequence of APAHRGRGGWTLNSAGYLLGPVLPVSSKADQGRKRDSALEILDLWKIIDGLPYSHSPRMT or a SPEXIN polypeptide having an amino acid sequence of NWTPQAMLYLKGAQ-NH2.

2. Use of a GALANIN family member for the preparation of a product for promoting delivery in mice, wherein, The GALANIN family member is a GALP polypeptide having an amino acid sequence of APAHRGRGGWTLNSAGYLLGPVLPVSSKADQGRKRDSALEILDLWKIIDGLPYSHSPRMT or a SPEXIN polypeptide having an amino acid sequence of NWTPQAMLYLKGAQ-NH2.

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