Application of homoarginine in preparation of products for improving placenta vascular development of mammals

Abstract

The invention discloses an application of homoarginine in preparation of a product for improving placental vascular development of mammals. The improvement of the placental vascular development of the mammals is specifically to increase the placental vascular density and / or up-regulate the expression of genes related to the placental vascular development. The invention also discloses an application of the homoarginine in preparation of a product for improving the reproductive performance of mammals. The invention further discloses a method for improving placenta vascular development of mammals and / or improving reproductive performance of mammals. The method comprises the step of feeding the mammals in the gestation period with homoarginine. The high arginine is used as the nutritional supplement for improving placenta vascular development of mammals (such as sows), the placenta vascular density can be effectively increased, the expression of related genes of vascular development is up-regulated, and the effects of increasing the placenta weight and the number of live piglets are achieved. According to the method, the problems of low litter size and high weak litter size can be solved, and theoretical guidance and data support are provided for enhancing placenta vascular development of mammals and improving economic benefits.

Description

Technical Field

[0001] The invention relates to the application of homoarginine in preparing a product for improving mammalian placental vascular development, and belongs to the field of precise nutritional regulation and reproduction. Background Art

[0002] As a "bridge" for the exchange of nutrients and gases between mother and fetus, the placenta plays an important regulatory role in the transport efficiency of nutrients during mammalian pregnancy. It enables the fetus to obtain oxygen and nutrients from the mother and excrete metabolic waste through blood circulation. Ensuring the normal development of placental blood vessels during mammalian pregnancy is a key link in maintaining maternal health and embryonic development. Placental vascular dysplasia, abnormalities, or dysfunction is an important factor leading to pregnancy complications such as intrauterine growth restriction, intrauterine fetal death, and preeclampsia, and affects fetal survival, growth, and health, and reduces reproductive performance.

[0003] Essential nutrients play an important regulatory role in the development of placental blood vessels in mammals during pregnancy. Homoarginine (hArg) is a pair of homologous amino acids with arginine (Arg). A first-in-human study has demonstrated that hArg supplementation is safe and well tolerated in humans. In addition, studies have found that hArg can serve as a substrate for NOS, promoting the production of nitric oxide (NO), thereby regulating vasodilation and vascular wall function, thereby affecting vascular development. NO plays a very important role in the vascular system, regulating vasodilation, blood flow and blood pressure. However, the application of hArg in human and animal research is still in its early stages, and there are currently no studies and reports on hArg and placental vascular development. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a new use of homoarginine - application in the preparation of products for improving the development of placental blood vessels in mammals. The present invention uses pigs as experimental animal models of mammals to conduct experiments on hArg regulating the development of placental blood vessels required for healthy pregnancy outcomes, deeply studies the mechanism of action of hArg, and provides scientific evidence for its function.

[0005] The present invention is achieved through the following technical solutions: Application of homoarginine in improving mammalian placental vascular development and application of homoarginine in preparing products for improving mammalian placental vascular development.

[0006] The improving of mammalian placental vascular development specifically includes: increasing placental vascular density and / or up-regulating the expression of genes related to placental vascular development.

[0007] Furthermore, the placental vascular development-related gene is selected from any one or more of PGF, VEGFA, ANGPT2 or eNOS.

[0008] Furthermore, the mammal is selected from pigs. Furthermore, the pig breed is Junmu No. 1.

[0009] Furthermore, the mammal is a female mammal in pregnancy, such as a pregnant sow.

[0010] Application of homoarginine in improving the reproductive performance of mammals and application of homoarginine in preparing products for improving the reproductive performance of mammals.

[0011] The improvement of mammalian reproductive performance specifically includes: increasing placental weight and / or increasing the number of live born piglets.

[0012] Furthermore, the mammal is selected from pigs. Furthermore, the pig breed is Junmu No. 1.

[0013] Furthermore, the mammal is a female mammal in pregnancy, such as a pregnant sow.

[0014] A method for enhancing placental vascular development and / or improving reproductive performance of a mammal is as follows: homoarginine is fed to a pregnant mammal.

[0015] Furthermore, the mammal is selected from pigs. Furthermore, the pig breed is Junmu No. 1.

[0016] Furthermore, homoarginine is added to the feed (daily diet) of pregnant sows. This method is easy to operate, saves time, and greatly avoids stress reactions on pregnant sows.

[0017] Furthermore, the added amount of homoarginine is 0.005% to 0.1% (weight percentage) of the pregnant sow feed, preferably 0.005%, 0.01% or 0.1%. The formulation of the pregnant sow feed meets the requirements of the National Research Council (NRC, 2012) for the nutritional standards of pregnant and lactating sows.

[0018] Furthermore, the time of feeding homoarginine is the whole gestation period, that is, the 0th to 114th day of the gestation period.

[0019] Furthermore, the homoarginine is L-homoarginine.

[0020] Furthermore, the purity of the homoarginine is 99%.

[0021] The present invention uses homoarginine as a nutritional supplement to improve the development of sow placental blood vessels. For the first time, by supplementing sows with 0.005% to 0.01% homoarginine during pregnancy, it was found that it can effectively increase the density of placental blood vessels, upregulate the expression of genes related to vascular development, increase the number of live piglets and placental weight, and solve the problems of low litter size and high number of weak piglets. It provides theoretical guidance and data support for enhancing the development of sow placental blood vessels and improving economic benefits in production.

[0022] The technical solution of the present invention has the following beneficial technical effects: (1) The present invention found through a full-term feeding experiment on pregnant sows that dietary supplementation with high arginine increased the levels of maternal serum TP, ALB and IgA and antioxidant capacity, increased the anti-inflammatory level of the placenta; improved and upregulated the placental vascular density and the expression of genes related to placental vascular development (such as PGF, VEGFA, ANGPT2 and eNOS), and effectively increased the number of live piglets and placental weight.

[0023] (2) The present invention improves the reproductive performance of sows in the form of nutritional supplements, and has the advantages of being economical, efficient, flexible, safe and widely applicable. It avoids potential problems such as veterinary drug residues and withdrawal periods in mammals such as pigs, and ensures the quality and safety of mammal products such as pork. Moreover, the method of the present invention is safe and harmless to the mother itself.

[0024] (3) The present invention is of great significance in promoting the development of placental blood vessels and thus improving the reproductive performance of mammals. It provides theoretical guidance and data support for developing new nutritional regulation technologies to improve the reproductive performance of mammals and improve economic benefits.

[0025] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : Schematic diagram of the effect of high arginine on the reproductive capacity of sows, where A: litter size; B: number of live piglets born; C: live piglet birth weight; D: live piglet litter weight; E: placenta weight. T1 (0.005%): 0.005% high arginine group; T2 (0.01%): 0.01% high arginine group; T3 (0.1%): 0.1% high arginine group, the same below.

[0027] Figure 2 : Schematic diagram of the effect of high arginine on the antioxidant capacity of sows, where A: serum total antioxidant capacity; B: serum superoxide dismutase activity C: serum glutathione concentration.

[0028] Figure 3: Schematic diagram of the effect of high arginine on the expression of anti-inflammatory cytokine genes in sow placenta, where A: TNF-; B: IFN-; C: IL-6; D: IL-8.

[0029] Figure 4 : Schematic diagram of HE staining of the placenta in each treatment group, where Control refers to the control group.

[0030] Figure 5 :Schematic diagram of the effect of homoarginine on vascular density in the sow placenta.

[0031] Figure 6 : Schematic diagram of the effect of high arginine on the gene expression of sow placental vascular development, where A: VEGFA; B: ANGPT2; C: PGF; D: eNOS. DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0033] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0034] The homoarginine used in the present invention was purchased from Xi'an Yunhe Biotechnology Co., Ltd. and is food grade with a purity of 99%.

[0035] The gestation sow diet (gestation diet) prepared by the present invention meets the requirements of the National Research Council (NRC, 2012) for the nutrient standards of gestation and lactation sows, and its components and nutrient levels are shown in Table 1. Each kilogram of the diet provides: vitamin A4125 IU, vitamin D3 881 IU, vitamin E 66 IU, vitamin K 1.10 mg, vitamin B1 1.30 mg, vitamin B2 4.25 mg, vitamin B6 1.40 mg, vitamin B12 25.50 mg, niacin 27.80 mg, folic acid 1.30 mg, biotin 0.30 mg, manganese 25 mg, zinc 100 mg, iron 80 mg, copper 10 mg, iodine 0.22 mg, and selenium 0.15 mg.

[0036]

[0037] Effects of dietary homoarginine supplementation during experimental pregnancy on placental vascular development and reproductive performance in sows 1. Experimental animals and experimental methods 60 Junmu No. 1 sows with similar body weight and parity were selected. They were randomly assigned to 4 diet groups (1 control group and 3 treatment groups), with 15 replicates in each group and 1 pig in each replicate. The control group was fed with a pregnancy diet without homoarginine, and the treatment groups were fed with a pregnancy diet with 0.005% homoarginine, a pregnancy diet with 0.01% homoarginine, and a pregnancy diet with 0.1% homoarginine, respectively. Sows were fed regularly every day at 08:00 and 14:00, respectively, and had free access to drinking water. This experiment started from day 0 of pregnancy to day 114 of farrowing. During the experiment, immunization procedures, routine management, and cleaning and disinfection were carried out according to the standard procedures of large-scale farms.

[0038] Blood was collected from sows on the 60th and 90th days of pregnancy, about 5 mL, and centrifuged at 4°C and 3000 r / min for 15 min. Serum was collected and stored at -80°C for the detection of biochemical and antioxidant indices. Blood was collected from sows by the anterior vena cava method. Subsequently, on the day of delivery, the sows weighed the placenta immediately after giving birth to the fetus and delivering the placenta, and 5 2 cm × 2 cm placental specimens were collected at about 5 cm around the junction of the umbilical cord and placenta within 30 min. After the placental tissue was rinsed with saline, the surface liquid was blotted with clean filter paper and stored in 4% paraformaldehyde solution and -80°C refrigerator for calculation of vascular distribution per unit area of ​​the placenta, measurement of placental inflammation level and expression of vascular development-related genes such as PGF, VEGFA, ANGPT2 and eNOS. At the same time, the reproductive performance of sows after delivery (litter size, number of live piglets, live piglet birth weight, live piglet litter weight and placenta weight, etc.) was recorded.

[0039] The detection of antioxidant indexes in pregnant sow serum was carried out using kits purchased from Nanjing Jiancheng Bioengineering Institute (China), and all operations and calculations were performed according to the kit instructions.

[0040] The vascular distribution of sow placental tissue was determined by hematoxylin-eosin (HE) staining. The placental tissue fixed with 4% paraformaldehyde solution was dehydrated in ethanol and xylene, then embedded in paraffin and sliced ​​continuously with a thickness of 4 μm. The slices were spread out in warm water and attached to a glass slide to dry. After drying, the slices were dewaxed in xylene twice, 5 min each time, and then placed in anhydrous ethanol for 2 min, 95% ethanol for 1 min, 80% ethanol for 1 min, and 75% ethanol for 1 min. Then, conventional HE staining was performed, including hematoxylin staining for 5 min, water washing for 3 min, 1% hydrochloric acid ethanol solution differentiation for 30 s, soaking in water for 10-15 min, 0.5% eosin solution staining for 2-3 min, and finally washed with water, wiped dry and dehydrated in ethanol. The slices were sealed and flattened with xylene transparent gum. The 100× slices were observed under an optical microscope to calculate the vascular distribution per unit area.

[0041] Placental inflammatory factors and vascular development-related genes were detected by RT-qPCR. Total RNA was isolated from cells using a kit from Takara (Japan). The concentration and purity of RNA were detected using an Eppendorf ultramicronucleic acid analyzer. The A260 / 280 ratio of 1.8 to 2.0 indicated acceptable RNA purity. Complementary DNA (cDNA) was generated by reverse transcription using a fast quantitative RT kit containing gDNase (Thermo Fisher, USA). RT-qPCR analysis was performed using a SuperReal PreMix Plus (SYBR Green) kit (Thermo Fisher, USA). A 20 μL standard reaction system containing three replicate wells was set up for each sample on a 96-well plate. The reaction procedure included a preliminary denaturation cycle at 95°C for approximately 15 minutes, followed by 40 thermal cycles of denaturation at 95°C for 10 seconds and annealing at 60°C for 32 seconds. The fluorescent signal was captured during the entire annealing phase. Gene sequences were obtained from GenBank. β-actin was selected as an internal reference gene by designing primers using conserved regions. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd., China. Table 2 shows the primer sequences and 2 -ΔΔCt Methods The relative mRNA expression of target genes was calculated.

[0042]

[0043] Numerical data are expressed as "mean ± standard deviation". One-way analysis of variance was performed using IBM SPSS Statistics 26.0 to analyze the data. Duncan's multiple comparisons were performed based on significant differences to examine the significance of each group of data (P < 0.05). The criterion for judging the significance of the difference was P < 0.05 ( * or #).

[0044] 2. Test results 2.1 Effects of feeding high arginine during pregnancy on reproductive performance of sows. Figure 1 shown.

[0045] like Figure 1 As shown in the figure, 0.005% hArg had a trend towards increasing total litter size compared to the control group, but the difference was not significant. At the same time, 0.005% hArg significantly increased the number of live piglets born and placenta weight. It is worth noting that the high dose of hArg (0.1% hArg) reduced the number of litters, the number of live piglets born, and the weight of live piglets.

[0046] 2.2 Effects of feeding high arginine during pregnancy on blood biochemical parameters of sows. The results are shown in Table 3.

[0047]

[0048] Note: *, P<0.05; **, P<0.01.

[0049] As shown in Table 3, compared with the control group, at 60 and 90 days of pregnancy, 0.005% hArg significantly increased the levels of TP, ALB and IgA in the serum of sows, 0.01% hArg significantly increased the levels of TP and IgA in the serum of sows, and 0.1% hArg only had a significant effect on the content of IgA in the serum of sows. All experimental groups had no significant effect on the content of TC and TG in the serum of sows.

[0050] 2.3 Effect of feeding high arginine during pregnancy on the antioxidant capacity of sows. The results are shown in Table 4. Figure 2 shown.

[0051]

[0052] Note: * , P<0.05; ** , P<0.01.

[0053] As shown in Table 4, Figure 2As shown in the results, compared with the control group, the addition of 0.005%, 0.01% and 0.1% hArg significantly increased the concentration of T-AOC in the serum of sows at 60 days of gestation. In addition, 0.005% and 0.01% hArg also significantly increased the concentration of T-AOC in the serum at 90 days of gestation. At the same time, 0.01% hArg significantly increased the activity of SOD in the serum at 60 days of gestation, but at the 90th day of gestation, all three hArg treatment groups showed a significant effect of increasing the activity of SOD in the serum. The addition of 0.005% hArg to the diet significantly increased the concentration of GSH in the serum at 60 and 90 days of gestation, while 0.01% and 0.1% had no significant effect on the control group.

[0054] 2.4 Effect of feeding high arginine during pregnancy on the expression of anti-inflammatory cytokine genes in sow placenta. Figure 3 shown.

[0055] like Figure 3 As shown in the results, 0.005% and 0.01% hArg significantly reduced the gene expression of proinflammatory cytokines IFN-, IL-6, and IL-8 in pig placental tissue. In addition, 0.005% hArg also significantly reduced the gene expression of TNF- in pig placental tissue. However, although 0.1% hArg significantly downregulated the gene expression of IL-6 and IL-8, it significantly upregulated the mRNA expression of TNF-.

[0056] 2.5 Effect of feeding high arginine during pregnancy on placental vascular development in sows. Figure 4 , Figure 5 , Figure 6 shown.

[0057] The development of placental blood vessels directly affects the oxygen supply and nutrition of the fetus, and plays a decisive role in the growth and development of the fetus. Figure 4 , Figure 5 As shown in Figure 2, 0.005% and 0.01% hArg supplementation significantly increased the vascular density of the sow placenta, while 0.1% hArg had no significant effect on the vascular density of the sow placenta. Figure 6 As shown in the figure, 0.005% hArg significantly upregulated the expression of VEGFA, ANGPT, PGF and eNOS, among which the mRNA expression of angiopoietin ANGPT2 was dose-dependent with the added level of hArg.

[0058] 3. Conclusion By adding homoarginine to the sow's diet throughout pregnancy, the experimental results showed that homoarginine can increase the number of live piglets and maternal placental weight, increase the levels of TP, ALB and IgA in maternal serum and antioxidant capacity, increase the placental anti-inflammatory level, improve and upregulate the placental vascular density, and the expression of placental vascular development-related genes such as PGF, VEGFA, ANGPT2 and eNOS.

[0059] Therefore, the method of the present invention is beneficial to promoting the health of mammals, improving placental development, and will not cause adverse effects on the maintenance of maternal pregnancy, thereby improving the reproductive performance of mammals.

[0060] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. Application of homoarginine in the preparation of products for improving the development of placental blood vessels in mammals, and application of homoarginine in improving the development of placental blood vessels in mammals.

2. The use according to claim 1, characterized in that: The improving of mammalian placental vascular development specifically includes: increasing the placental vascular density and / or up-regulating the expression of genes related to placental vascular development.

3. The use according to claim 2, characterized in that: The placental vascular development-related gene is selected from any one or more of PGF, VEGFA, ANGPT2 and eNOS.

4. The use according to claim 1, 2 or 3, characterized in that: The mammal is selected from pigs.

5. Use of homoarginine in the preparation of a product for improving the reproductive performance of mammals, or in improving the reproductive performance of mammals.

6. The use according to claim 5, characterized in that: The improvement of mammalian reproductive performance specifically includes: increasing placental weight and / or increasing the number of live born piglets.

7. The use according to claim 5, characterized in that: The mammal is selected from pigs.

8. A method for improving placental vascular development and / or reproductive performance of mammals, characterized in that: Feeding homoarginine to pregnant mammals.

9. The method according to claim 8, characterized in that: The mammal is selected from pigs.

10. The method according to claim 9, characterized in that: Adding homoarginine to gestating sow feed; Or: the feeding time of high arginine is the whole period of pregnancy; Or: the added dosage of the homoarginine is 0.005% to 0.1% of the pregnant sow feed.

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