Lactobacillus murine Mafic1501 and application thereof in improving placenta function of matrix with limited intrauterine growth and / or embryonic development with limited intrauterine growth

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus murine Mafic1501 and application thereof in improving the placenta function of a matrix with limited intrauterine growth and / or the development of an embryo with limited intrauterine growth. According to the present invention, it is found that the maternal placenta function and the fetal development can be improved by adding the Lactobacillus murinus Mafic1501 to the maternal source, such that the Lactobacillus murinus Mafic1501 and the application thereof are provided, the Lactobacillus murinus Mafic1501 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.32163. The invention further provides the application of the Lactobacillus murinus Mafic1501, the invention provides an effective nutrition regulation and control scheme for relieving the adverse pregnancy outcome with limited intrauterine growth.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Lactobacillus murinus Mafic1501 and application thereof in improving maternal placental function of intrauterine growth restriction and / or intrauterine growth restricted embryonic development. Background Art

[0002] Intrauterine growth restriction (IUGR), also known as fetal growth restriction (FGR), refers to the impaired growth and development of the mammalian embryo / fetus during pregnancy (Wang et al., 2017). The prevalence of IUGR is not low. IUGR can change the health of the mother, increase the risk of perinatal mortality and morbidity of the fetus, cause neonatal immune dysfunction and intestinal barrier damage, and bring short-term / long-term adverse health consequences (Huang et al., 2019; Tao et al., 2019). It can be seen that it is imperative to reduce the incidence of intrauterine growth restriction and its adverse pregnancy outcomes. The placenta is the link between nutrient supply and embryonic growth during pregnancy, and plays a key role in immune system acceptance, nutrient delivery, and metabolite excretion (Sharma et al., 2016; Woods et al., 2018). Studies have shown that IUGR is related to placental inefficiency. Reduced sinusoidal area, inflammatory response, and oxidative damage can all cause placental dysfunction, leading to the occurrence of IUGR. Therefore, establishing a nutritional regulation program to improve maternal placental function is one of the keys to improving intrauterine growth restriction and its adverse pregnancy outcomes.

[0003] Lactic acid bacteria are typical probiotics that can regulate host health by improving the balance of intestinal microorganisms. Intervention during pregnancy can affect the structure of the maternal flora and the growth and development of the fetus. Studies have shown that adding rhamnosus to the diet of pregnant and lactating women can reduce maternal blood sugar and insulin concentrations (Laitinen et al, 2009). In addition, by supplementing pregnant women with lactobacilli, plasma glutathione, erythrocyte glutathione reductase and other indicators can be increased, which can alleviate oxidative stress and premature birth, low birth weight and other problems (Asemi et al., 2013). Among the many lactic acid bacteria, Lactobacillus murinus ( L. murinus ) can colonize in the intestine and relieve neonatal necrotizing enterocolitis (Isani et al., 2018). However, its effect and mechanism of improving maternal placental function and intrauterine growth restriction have not been reported. Summary of the invention

[0004] One of the purposes of the present invention is to provide a novel method for improving maternal placental function and / or intrauterine growth restricted embryo development.

[0005] The present invention provides Lactobacillus murinus ( L. murinus ) Mafic1501, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC NO. 32163.

[0006] Intrauterine growth restriction (IUGR) increases the risk of perinatal mortality and morbidity, causes neonatal immune dysfunction and intestinal barrier damage, and brings short-term / long-term adverse health consequences. Based on this, the present invention uses IUGR pregnant mice and their fetuses as test subjects and finds a strain of Lactobacillus murinus ( L. murinus ) Mafic1501, from the perspective of mother-child integration ("mother-placenta-fetus"), analyzed L. murinus The effects and mechanisms of improving placental function and fetal development provide an effective new nutritional regulation solution to alleviate the adverse pregnancy outcomes of intrauterine growth restriction.

[0007] Specifically, the present invention establishes an intrauterine growth restricted pregnant mouse model, and through an animal feeding experiment of supplementing Lactobacillus murine Mafic1501 during pregnancy, combined with a comprehensive analysis of maternal litter performance, plasma immunity and oxidation indicators, placental efficiency and sinusoidal area, and fetal intestinal immunity and barrier function, deeply explores the effect and mechanism of Lactobacillus murine in regulating intrauterine growth restricted placental function and fetal development, fills the application gap of Lactobacillus murine in improving maternal and fetal nutrition and health, and provides a theoretical basis for the development of special probiotics to alleviate the adverse pregnancy outcomes of intrauterine growth restriction.

[0008] The present invention also provides a bacterial agent, which contains the above-mentioned Lactobacillus murinus ( L. murinus )Mafic1501.

[0009] The bacterial agent of the present invention can be a liquid bacterial agent or a solid bacterial agent, which contains Lactobacillus murinus ( L. murinus ) In addition to Mafic1501, other strains with the same or different functions may also be included.

[0010] The present invention also provides the above-mentioned Lactobacillus murinus ( L. murinus )Application of Mafic1501 or bacterial agent in any of the following aspects: Improve placental function in mothers with intrauterine growth restriction and / or fetal (intestinal) development in intrauterine growth restricted women; Improve the litter performance of intrauterine growth restricted maternal mice and / or the body weight of intrauterine growth restricted fetal mice; Alleviate inflammation and / or oxidative stress in intrauterine growth restricted maternal mice; Improve the efficiency of placentation in intrauterine growth restricted maternal mice; Improve the intestinal barrier function of intrauterine growth restricted fetal mice; Improve the development of intestinal immune function in intrauterine growth restricted fetal mice.

[0011] The present invention also provides a method for improving maternal placental function and / or fetal development of intrauterine growth restriction, wherein the above-mentioned Lactobacillus murinus ( L. murinus )Mafic1501 or bacterial agent.

[0012] The applications and methods of the present invention are for purposes other than disease diagnosis or treatment, and can be used in pharmaceutical research and pathogenic mechanism research.

[0013] The present invention also provides an oral preparation for improving maternal placental function of intrauterine growth restriction and / or intrauterine growth restricted embryonic development, which comprises the above-mentioned Lactobacillus murinus ( L. murinus )Mafic1501 or bacterial agent.

[0014] Supplementation of Lactobacillus murinus Mafic1501 during pregnancy can significantly improve the litter performance of pregnant mice, improve the efficiency and function of the placenta, promote the development of the fetal intestine, and thus alleviate the adverse pregnancy outcomes caused by intrauterine growth restriction. The strain of the present invention can be used to develop safe and effective maternal and fetal nutritional additive products, form a nutritional regulation program to improve maternal health, improve placental efficiency and function, promote fetal development, and reduce adverse pregnancy outcomes caused by intrauterine growth restriction.

[0015] The beneficial effects of the present invention are at least: The present invention finds that maternal addition of Lactobacillus murine Mafic1501 can improve maternal placental function and fetal development, and provides an effective nutritional regulation program for alleviating adverse pregnancy outcomes caused by intrauterine growth restriction. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The colony (A), morphology (B) and growth curve (C) of Lactobacillus murinus.

[0017] Figure 2 Schematic diagram of the construction of the intrauterine growth restriction model and the design of the Lactobacillus murinus intervention experiment.

[0018] Figure 3 It is an index of maternal weight and litter performance. Among them, A is maternal weight, B is litter size, C is litter weight, and D is fetal weight.

[0019] Figure 4 Indicators of serum inflammation and oxidative stress in maternal mice. A is the result of IL-1β, B is the result of TNF-α, C is the result of IL-6, D is the result of T-AOC, E is the result of GSH-Px, and F is the result of MDA.

[0020] Figure 5 These are placenta indicators of intrauterine growth restricted pregnant mice. A is the statistical result of placenta weight, B is the statistical result of fetal mouse / placenta ratio, C is the observation result of placental tissue, the second row of pictures is the enlarged picture of the yellow frame area in the first row of photos, and D is the statistical result of blood sinus area.

[0021] Figure 6 It is an indicator of intestinal development in fetal mice with intrauterine growth restriction. Among them, A is the observation result of the jejunal villi morphology of fetal mice, B is Claudin-1 The relative expression of the gene, C is Occludin The relative expression of the gene, D is ZO-1 Relative gene expression.

[0022] Figure 7 The intestinal immune indexes of intrauterine growth restricted fetal mice. Among them, A is the polarization status of fetal intestinal macrophages, B is the positive rate of CD11c, C is the positive rate of CD206, and D is IRF5 The relative expression of the gene, E is IRF4 The relative expression of the gene, F is IL-1β The relative expression of the gene, G is TNF-α The relative expression of the gene, H is IL-4Ra The relative expression of the gene, I IL- 10 Relative gene expression.

[0023] In each figure (if any), represents P < 0.05, represent P <0.01. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0025] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0026] Example 1 Lactobacillus murinus Lactobacillus murinus ( L. murinus )Screening and identification of Mafic1501

[0027] (1) Lactobacillus murinus L. murinusIsolation and seed preservation of Mafic1501

[0028] Lactobacillus murinus was isolated from the feces of ICR mice, which were raised in the Experimental Animal Center of the Feed Industry Center of the Ministry of Agriculture and Rural Affairs. The isolation method was as follows (plate coating and streaking): 1 ICR mouse feces sample was taken and graded diluted with sterile PBS buffer. 10 -1 -10 -3 100 μL of the dilution solution was evenly spread on the MRS agar plate culture medium. After anaerobic culture at 37°C for 24 h, single colonies were randomly selected on the plate according to the colony morphology (size, color, etc.), numbered, and inoculated on the MRS agar plate culture medium for streak purification and anaerobically cultured; the purified single colony was picked and inoculated into the MRS liquid culture medium tube that had been previously inflated with CO2. After anaerobic culture at 37°C for 24 h, 50% sterile glycerol was used to preserve the strain and 1 mL was taken for strain identification and seed preservation.

[0029] (2) Lactobacillus murinus L. murinus Identification and preservation of Mafic1501

[0030] Take the strain L. murinus Mafic1501 bacterial liquid, total bacterial DNA was extracted, PCR amplified using bacterial universal primers (27F and 1492R), and 16S rRNA gene sequencing was performed for identification. The strain was compared with the NCBI database and was determined to be Lactobacillus murinus ( L. murinus The strain was named Lactobacillus murinus Mafic1501, which was deposited on October 10, 2024 at the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified as Lactobacillus murinus Lactobacillus murinus , the deposit number is CGMCC No. 32163.

[0031] (3) Colony, morphology and growth curve of Lactobacillus murinus

[0032] L. murinus The diameter of a single colony of Mafic1501 is about 1 mm, and the color is white and opaque. The surface of the colony is slightly convex and the edges are irregular (see Figure 1 A in the figure); Gram staining and optical microscopy, L. murinus Mafic1501 is a Gram-positive bacterium with a slender rod shape, no flagella, and no motility (see Figure 1 B); MRS liquid culture and determination of OD600 values ​​at different time points to obtain its growth curve, L. murinus Mafic1501 entered the logarithmic growth phase at 4 h and the plateau phase at 12 h (see Figure 1 C in.

[0033] Example 2 Efficacy verification of Lactobacillus murinus Mafic1501

[0034] Experimental design and animal husbandry: Eighteen 8-week-old ICR female mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) were selected for the experiment. One male and two females were put into the same cage at 20:00 on the same day. The vaginal plugs of the female mice were checked at 8:00 the next day. The presence of vaginal plugs was recorded as gestational day 0.5 (G0.5). Figure 2 As shown in the figure, pregnant mice were divided into three groups: control group (CON), intrauterine growth restriction group (IUGR) and intrauterine growth restriction + Lactobacillus murinus group (IUGR + LM); in the first and second trimesters of pregnancy (G0.5-G9.5), all pregnant mice were fed the same basal diet (normal diet, ND, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.); in the second and third trimesters of pregnancy (G10.5-G18.5), pregnant mice in the CON group were fed with a basal diet, and pregnant mice in the IUGR group and IUGR + LM group were fed with a low protein diet (low protein diet, LP, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) to establish the IUGR pregnant mouse model; pregnant mice in the CON group and IUGR group were gavaged with PBS, and pregnant mice in the IUGR + LM group were gavaged with Lactobacillus murinus Mafic1501 (LM, 2×10 9 CFU / d), with 6 replicates in each group, and pregnant female mice were housed in single cages.

[0035] Test results: (1) Supplementation of Lactobacillus murinus during pregnancy improves litter performance and pup weight in intrauterine growth restricted rats

[0036] At the end of the experiment, the weight of the mother mice, the number of pups born, the weight of the fetuses and the weight of the litter were counted, and the performance of the mother mice was calculated. Figure 3 As shown, in the intrauterine growth restriction model, supplementation with Lactobacillus murinus during pregnancy had no significant effect on maternal body weight and litter size (see Figure 3 A and B in P >0.05, and significantly increased fetal weight and litter weight (see Figure 3 C, D in; P <0.05), thereby improving the litter performance of intrauterine growth restricted rats.

[0037] (2) Supplementation of Lactobacillus murinus during pregnancy alleviates inflammation and oxidative stress in rats with intrauterine growth restriction

[0038] At the end of the experiment, serum was collected from the mother mice for determination of inflammation and oxidative stress indicators. Figure 4As shown, in the intrauterine growth restriction model, supplementation with Lactobacillus murinus during pregnancy significantly reduced the levels of proinflammatory cytokines IL-1β, TNF-α, and IL-6 in maternal serum (see Figure 4 AC in; P <0.05); In addition, supplementation with Lactobacillus murinus during pregnancy significantly increased the total antioxidant capacity (T-AOC, see Figure 4 D in; P <0.05) and glutathione peroxidase (GSH-Px, see Figure 4 E in; P <0.05) level and reduce serum malondialdehyde level (MDA, see Figure 4 F in; P <0.05). The above results indicate that supplementation of Lactobacillus murinus during pregnancy alleviates inflammation and oxidative stress in intrauterine growth restricted rats.

[0039] (3) Supplementation of Lactobacillus murinus during pregnancy improves placental efficiency in intrauterine growth restricted mice

[0040] At the end of the experiment, the female mice were slaughtered and the placenta was weighed to calculate the placental efficiency. The placental tissue was fixed in 4% paraformaldehyde for sectioning, H&E staining and observation of blood flow area to comprehensively evaluate the placental development. Figure 5 As shown, supplementation with Lactobacillus murinus during pregnancy had no significant effect on placental weight (see Figure 5 A in; P> 0.05), and significantly increased the fetal / placenta ratio in intrauterine growth restricted rats (see Figure 5 B in; P <0.05) and sinusoidal area (see Figure 5 C, D in; P <0.05), thereby improving the efficiency of the placenta of pregnant mice and promoting maternal-fetal nutrient transport.

[0041] (4) Maternal supplementation with Lactobacillus murinus improves intestinal barrier function in intrauterine growth restricted fetal mice

[0042] At the end of the experiment, the mother mice were slaughtered and the fetal jejunal tissue was fixed with 4% paraformaldehyde for sectioning, H&E staining and observation of villus morphology and structure. Another jejunal tissue sample was frozen in liquid nitrogen for RT-qPCR to measure the expression of intestinal tight junction genes and comprehensively evaluate the fetal intestinal development. Figure 6 As shown, maternal supplementation with Lactobacillus murinus improved the intestinal villus morphology of intrauterine growth restricted fetal mice (see Figure 6 A), and improve intestinal tight junctions Claudin-1 , Occludin and ZO-1 Gene expression (see Figure 6 BD in; P<0.05).

[0043] (5) Maternal supplementation with Lactobacillus murinus improves the development of intestinal immune function in intrauterine growth restricted fetal mice

[0044] At the end of the experiment, the mother mice were slaughtered and the fetal jejunal tissue was fixed with 4% paraformaldehyde for sectioning, immunohistochemical staining and observation of macrophage polarization. Another jejunal tissue sample was frozen in liquid nitrogen, and total RNA was extracted from the tissue for RT-qPCR to measure the expression of intestinal macrophage polarization markers and immune cytokines, and to comprehensively evaluate the development of fetal intestinal immune function. Figure 7 As shown in Figure 2, maternal supplementation with Lactobacillus murinus improved the polarization of intestinal macrophages in intrauterine growth restricted fetal mice and inhibited the differentiation of silent macrophages into pro-inflammatory macrophages (CD11c + , see Figure 7 A, B in; P <0.05, but had no effect on the polarization of anti-inflammatory macrophages (CD206 + , see Figure 7 A, C in; P >0.05); at the same time, maternal supplementation with Lactobacillus murinus significantly reduced the markers of pro-inflammatory macrophages in the intestine of intrauterine growth restricted fetal mice IRF5 Gene expression (see Figure 7 D in; P <0.05), while increasing pro-inflammatory macrophage markers IRF4 Gene expression (see Figure 7 E in; P <0.05); In addition, maternal supplementation with Lactobacillus murinus inhibited the intestinal proinflammatory cytokines in intrauterine growth restricted fetal mice IL-1β and TNF-α Gene expression (see Figure 7 F, G in; P <0.05), and increase the anti-inflammatory cytokine receptors IL-4Ra Gene expression (see Figure 7 H in; P <0.05), while the inhibitory cytokines IL-10 There was no significant effect on gene expression (see Figure 7 I in; P >0.05), thereby promoting the development of the immune function of fetal mice.

[0045] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Lactobacillus murinus L. murinus ) Mafic1501, characterized in that, It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC NO.32163.

2. A bacterial agent, characterized in that Containing the Lactobacillus murinus according to claim 1 ( L. murinus )Mafic1501.

3. The Lactobacillus murinus of claim 1 ( L. murinus ) Use of Mafic1501 or the bacterial agent described in claim 2 in improving maternal placental function and / or intrauterine growth restricted embryo development.

4. The Lactobacillus murinus of claim 1 ( L. murinus ) Use of Mafic1501 or the bacterial agent described in claim 2 in improving the litter performance of intrauterine growth restricted mother mice and / or the weight of intrauterine growth restricted fetal mice.

5. The Lactobacillus murinus of claim 1 ( L. murinus ) Use of Mafic1501 or the bacterial agent described in claim 2 in alleviating inflammation and / or oxidative stress in intrauterine growth restricted female mice.

6. The Lactobacillus murinus of claim 1 ( L. murinus ) Use of Mafic1501 or the bacterial agent described in claim 2 in improving the efficiency of placenta in intrauterine growth restricted mice.

7. The Lactobacillus murinus of claim 1 ( L. murinus ) Use of Mafic1501 or the bacterial agent described in claim 2 in improving the intestinal barrier function of intrauterine growth restricted fetal mice.

8. The Lactobacillus murinus of claim 1 ( L. murinus ) Use of Mafic1501 or the bacterial agent described in claim 2 in improving the development of intestinal immune function in fetal mice with intrauterine growth restriction.

9. A method for improving maternal placental function and / or intrauterine growth restricted fetal development, characterized in that: Taking the Lactobacillus murinus described in claim 1 during pregnancy L. murinus ) Mafic1501 or the bacterial agent described in claim 2.

10. An oral preparation for improving maternal placental function and / or intrauterine growth restricted embryonic development, characterized in that: comprising the Lactobacillus murinus described in claim 1 ( L. murinus ) Mafic1501 or the bacterial agent described in claim 2.

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