Lactobacillus murinus mafic1501 and its use in improving placental function in intrauterine growth restricted mothers and / or development of intrauterine growth restricted embryos

Abstract

The present application relates to the field of microbial technology, and specifically discloses Lactobacillus murinus Mafic1501 and its application in improving placental function of intrauterine growth restriction maternal and / or embryo development of intrauterine growth restriction. L. murinus The present application finds that adding Lactobacillus murinus Mafic1501 to the mother can improve the placental function of the mother and the development of the fetus, and further provides a strain of Lactobacillus murinus (Mafic1501) and its application. The Lactobacillus murinus Mafic1501 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.32163. It provides an effective nutritional control scheme for alleviating the adverse pregnancy outcomes of intrauterine growth restriction.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular, to Lactobacillus murinus Mafic1501 and its application in improving placental function of intrauterine growth restriction mother and / or development of intrauterine growth restriction embryo. BACKGROUND

[0002] Intrauterine growth restriction (IUGR), also known as fetal growth restriction (FGR), refers to impaired growth and development of mammalian embryo / fetus during pregnancy (Wang et al., 2017). The prevalence of IUGR is not low. IUGR can change the health status of the mother, increase the risk of perinatal mortality and morbidity of the fetus, cause immune dysfunction and intestinal barrier damage of the newborn, and bring short-term / long-term adverse health consequences (Huang et al., 2019; Tao et al., 2019). Therefore, it is imperative to reduce the incidence of intrauterine growth restriction and its adverse pregnancy outcomes. The placenta is the link of nutrient supply and embryo growth during pregnancy, and plays a key role in immune system acceptance, nutrient transport, and metabolite discharge (Sharma et al., 2016; Woods et al., 2018). Studies have shown that IUGR is associated with low placental efficiency, and reduced blood sinus area, inflammatory response and oxidative damage can all cause placental dysfunction, which in turn leads 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, which can regulate host health by improving intestinal microbial balance, and intervention during pregnancy can affect maternal flora structure and fetal growth and development. Studies have shown that the addition of Lactobacillus rhamnosus in the diet of pregnant and lactating women can reduce maternal blood glucose, insulin concentration, etc. (Laitinen et al, 2009). In addition, supplementing pregnant women with Lactobacillus can increase plasma glutathione, erythrocyte glutathione reductase, etc., and can alleviate oxidative stress and problems such as premature birth and low birth weight (Asemi et al., 2013). Among 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 in improving maternal placental function and intrauterine growth restriction have not been reported. L. L. murinus SUMMARY

[0004] ​One of the objectives of this invention is to provide a new method for improving placental function in intrauterine growth-restricted mothers and / or the development of intrauterine growth-restricted embryos.

[0005] This invention provides *Lactobacillus murineis* (… L. murinus Mafic1501 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession 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 leads to adverse short-term and long-term health consequences. Based on this, this invention uses IUGR-pregnant mice and their fetuses as experimental subjects to identify a strain of *Lactobacillus murineis* with good efficacy. L. murinus Mafic1501 analyzes the process from the perspective of maternal-fetal integration ("mother-placenta-fetus"). L. murinus The effects and mechanisms of improving placental function and fetal development provide a new and effective nutritional regulation strategy for alleviating adverse pregnancy outcomes caused by intrauterine growth restriction.

[0007] Specifically, this invention establishes an intrauterine growth restriction (IVR) mouse model and conducts an animal feeding experiment supplemented with Lactobacillus murineis Mafic1501 during pregnancy. Combined with a comprehensive analysis of maternal reproductive performance, plasma immune and oxidative indicators, placental efficiency and sinus area, and fetal intestinal immunity and barrier function, the invention delves into the effects and mechanisms of Lactobacillus murineis in regulating placental function and fetal development in IVR. This fills a gap in the application of Lactobacillus murineis in improving maternal and fetal nutrition and health, and provides a theoretical basis for developing a unique probiotic to alleviate adverse pregnancy outcomes associated with IVR.

[0008] The present invention also provides a bacterial agent containing the above-mentioned *Lactobacillus murineis* ( L. murinus Mafic1501.

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

[0010] This invention also provides the above-mentioned Lactobacillus murineis ( L. murinus The application of Mafic1501 or the microbial agent in any of the following aspects:

[0011] Improve placental function in mothers with intrauterine growth restriction and / or development of intrauterine growth restriction embryos (intestines);

[0012] Improve the reproductive performance of intrauterine growth-restricted female mice and / or the fetal weight of intrauterine growth-restricted mice;

[0013] alleviate inflammation and / or oxidative stress of IUGR maternal mice;

[0014] improve placental efficiency of IUGR maternal mice;

[0015] improve intestinal barrier function of IUGR fetal mice;

[0016] improve the development of intestinal immune function of IUGR fetal mice.

[0017] The application also provides a method for improving placental function of IUGR maternal and / or development of IUGR embryo, which comprises taking the above-mentioned Lactobacillus murinus (Mafic1501) or bacterial agent during pregnancy. L. murinus ) Mafic1501 or a bacterial agent.

[0018] The application and the method are both for non-disease diagnosis or treatment purposes, and can be used for pharmaceutical research and pathogenic mechanism research.

[0019] The application also provides an oral preparation for improving placental function of IUGR maternal and / or development of IUGR embryo, which comprises the above-mentioned Lactobacillus murinus (Mafic1501) or a bacterial agent. L. murinus ) Mafic1501 or a bacterial agent.

[0020] Supplementing Lactobacillus murinus Mafic1501 during pregnancy can significantly improve the litter performance of pregnant mice, improve placental efficiency and function, promote fetal intestinal development, and thus alleviate the adverse pregnancy outcomes caused by IUGR. The strain of the application can develop a safe and effective maternal and infant nutritional additive product, form a nutritional regulation scheme for improving maternal health, improving placental efficiency and function, and promoting fetal development, and reduce the adverse pregnancy outcomes caused by IUGR.

[0021] The application has at least the following beneficial effects:

[0022] The application finds that adding Lactobacillus murinus Mafic1501 to maternal sources can improve placental function of maternal and development of fetus, and provides an effective nutritional regulation scheme for alleviating adverse pregnancy outcomes of IUGR. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a colony (A), morphology (B) and growth curve (C) of Lactobacillus murinus.

[0024] Figure 2 It is a schematic diagram of IUGR model construction and Lactobacillus murinus intervention test design.

[0025] Figure 3 It is a maternal body weight and litter performance index. Among them, A is the maternal body weight, B is the number of litters, C is the litter weight, and D is the fetal body weight.

[0026] Figure 4The serum inflammation and oxidative stress indicators of the mother mouse. Among them, A is the detection result of IL-1β, B is the detection result of TNF-α, C is the detection result of IL-6, D is the detection result of T-AOC, E is the detection result of GSH-Px, and F is the detection result of MDA.

[0027] Figure 5 The placental indicators of the intrauterine growth restriction pregnant mouse. Among them, A is the placental weight statistical result, B is the fetal mouse / placenta ratio statistical result, C is the placental tissue observation result, the second row of pictures is the enlarged view of the yellow frame area in the first row of pictures, and D is the blood sinus area statistical result.

[0028] Figure 6 The intestinal development indicators of the intrauterine growth restriction fetal mouse. Among them, A is the fetal mouse jejunum villus morphology observation result, B is Claudin-1 the relative expression amount of the gene, C is Occludin the relative expression amount of the gene, D is ZO-1 the relative expression amount of the gene.

[0029] Figure 7 The intestinal immune indicators of the intrauterine growth restriction fetal mouse. Among them, A is the fetal mouse intestinal macrophage polarization condition, B is the positive rate of CD11c, C is the positive rate of CD206, D is IRF5 the relative expression amount of the gene, E is IRF4 the relative expression amount of the gene, F is IL-1β the relative expression amount of the gene, G is TNF-α the relative expression amount of the gene, H is IL-4Ra the relative expression amount of the gene, I is IL- 10 the relative expression amount of the gene.

[0030] In each figure (if any), represents P<0.05, represents P <0.01. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the following examples are given only to illustrate the present application and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and replacements to the present application without departing from the spirit and principles of the present application.

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels or prepared according to conventional methods in the art unless otherwise specified.

[0033] Example 1 Lactobacillus murinusLactobacillus murinus L. murinus Screening and identification of Mafic1501

[0034] (1) Lactobacillus murinus L. murinus Isolation and preservation of Mafic1501

[0035] Lactobacillus murinus was isolated from the feces of ICR mice raised in the Experimental Animal Center of the Feed Industry Center of the Ministry of Agriculture and Rural Affairs. The isolation method is as follows (plate streaking): take 1 particle of ICR mouse feces, gradient dilute with sterile PBS buffer, select 10 -1 -10 -3 100 μL of the diluent is uniformly coated on the MRS agar plate culture medium, and after 24 h of anaerobic culture at 37℃, according to the colony morphology (size, color, etc.), single colonies are randomly selected on the plate, numbered, inoculated on MRS agar plate culture medium, and anaerobic culture; pick the purified single colony, inoculate into the MRS liquid medium tube pre-filled with CO2, and after 24 h of anaerobic culture at 37℃, use 50% sterile glycerol to preserve the strain and take 1 mL for strain identification and preservation.

[0036] (2) Lactobacillus murinus L. murinus Identification and preservation of Mafic1501

[0037] Strain L. murinus Mafic1501 bacterial liquid, extract total bacterial DNA, use bacterial universal primers (27F and 1492R) for PCR amplification, then perform 16S rRNA gene sequencing identification, and compare in the NCBI database to determine the strain category as Lactobacillus murinus (Lactobacillus murinus). L. murinus The strain is named Lactobacillus murinus Mafic1501, which has been preserved in the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, postcode: 100101) on October 10, 2024, and classified as Lactobacillus murinus Lactobacillus murinus , with the preservation number CGMCC No. 32163.

[0038] (3) Colony, morphology and growth curve of Lactobacillus murinus

[0039] L. murinus The single colony of Mafic1501 is about 1 mm in diameter, white in color, opaque, with a slightly convex surface and irregular edges (see A in Figure 1 ); after Gram staining and observation under an optical microscope, L. murinus Mafic1501 is a gram-positive bacterium, long rod-shaped, non-flagellated, and non-motile (see​Figure 1 B) in Example 1; MRS liquid culture and determination of OD600 value at different time points to obtain their growth curves, L. murinus Mafic1501 entered logarithmic growth phase at 4 h and plateau phase at 12 h (see Figure 1 C) in Example 1.

[0040] Example 2. Efficacy verification of Lactobacillus murinus Mafic1501

[0041] Test design and animal feeding:

[0042] Eighteen 8-week-old ICR female mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) were selected for the test, and they were co-caged at 20:00 on the same day according to 1 male: 2 females. The next day at 8:00, the female mice with vaginal plugs were recorded as gestation day 0.5 (G0.5). If Figure 2 , the pregnant mice were divided into three groups: control group (CON), intrauterine growth restriction group (IUGR), and intrauterine growth restriction + Lactobacillus murinus group (IUGR+LM). During the middle of pregnancy (G0.5-G9.5), all pregnant mice were fed with the same normal diet (ND, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.). During the late pregnancy (G10.5-G18.5), the CON group was fed with the normal diet, and the IUGR and IUGR+LM groups were fed with a low-protein diet (LP, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) to construct the IUGR pregnant mouse model. The CON and IUGR groups were administered with PBS, and the IUGR+LM group was administered with Lactobacillus murinus Mafic1501 (LM, 2x10 9 CFU / d). There were 6 replicates in each group, and the pregnant mice were individually caged.

[0043] Test results:

[0044] (1) Lactobacillus murinus supplementation during pregnancy improves the litter performance and body weight of offspring of intrauterine growth restricted dams

[0045] At the end of the test, the body weight of the dams, the number of offspring, the body weight of the offspring, and the litter weight were calculated. As shown in Figure 3 , Lactobacillus murinus supplementation during pregnancy had no significant effect on the body weight of the dams and the number of offspring in the intrauterine growth restriction model (see Figure 3 A, B; P >0.05), but significantly improved the body weight of the offspring and the litter weight of the dams (see Figure 3 C, D; P <0.05), thereby improving the litter performance of intrauterine growth restricted dams.

[0046] (2) Supplementing pregnant mice with Lactobacillus murineis can alleviate inflammation and oxidative stress in intrauterine growth-restricted mice.

[0047] At the end of the experiment, serum was collected from the mother mice to measure indicators of inflammation and oxidative stress. Figure 4 As shown, in the intrauterine growth restriction model, supplementation with *Lactobacillus murineis* during pregnancy significantly reduced serum levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in maternal mice (see [link to study]). Figure 4 AC in the middle; P <0.05); In addition, supplementation with Lactobacillus murineis during pregnancy significantly increased the total antioxidant capacity (T-AOC) in maternal serum (see [reference needed]). Figure 4 D in P <0.05) and glutathione peroxidase (GSH-Px, see Figure 4 E in P It reduced serum malondialdehyde (MDA) levels to <0.05 and decreased serum malondialdehyde (MDA) levels (see [link]). Figure 4 F in P <0.05). These results indicate that supplementation with *Lactobacillus murineis* during pregnancy alleviates inflammation and oxidative stress in pregnant mice with intrauterine growth restriction.

[0048] (3) Supplementing with Lactobacillus murineis during pregnancy improves placental efficiency in intrauterine growth-restricted female mice.

[0049] At the end of the experiment, female mice were slaughtered and their placentas were collected and weighed to calculate placental efficiency. Placental tissue was fixed in 4% paraformaldehyde for sectioning, H&E staining, and observation of blood flow area to comprehensively evaluate placental development. Results are as follows: Figure 5 As shown, supplementation with Lactobacillus murineis during pregnancy has no significant effect on placental weight (see [reference]). Figure 5 A in P> 0.05), while significantly increasing the fetus / placenta ratio in intrauterine growth-restricted mothers (see Figure 5 B in the middle; P <0.05) and sinus area (see Figure 5 C, D; P <0.05), thereby improving placental efficiency in pregnant mice and promoting maternal-fetal nutrient transport.

[0050] (4) Maternal supplementation with Lactobacillus murineis enhances intestinal barrier function in intrauterine growth-restricted fetal rats.

[0051] At the end of the experiment, female mice were slaughtered and fetal jejunal tissue was fixed in 4% paraformaldehyde for sectioning, H&E staining, and observation of villus morphology. Separate jejunal tissue samples were frozen in liquid nitrogen for RT-qPCR to determine the expression levels of intestinal tight junction genes, thus comprehensively evaluating the fetal intestinal development status. Figure 6 As shown, maternal supplementation with *Lactobacillus murineis* improves the morphology of intestinal villi in intrauterine growth-restricted fetal rats (see [reference]). Figure 6(A) and improve intestinal tight junctions. Claudin-1 , Occludin and ZO-1 Gene expression levels (see) Figure 6 BD in the middle; P <0.05).

[0052] (5) Maternal supplementation with Lactobacillus murineis enhances the development of intestinal immune function in intrauterine growth-restricted fetal rats.

[0053] At the end of the experiment, female mice were slaughtered and fetal jejunal tissue was fixed in 4% paraformaldehyde for sectioning, immunohistochemical staining, and observation of macrophage polarization. Separately, jejunal tissue samples were frozen in liquid nitrogen, and total RNA was extracted for RT-qPCR to determine intestinal macrophage polarization markers and the expression levels of immune cytokines, comprehensively evaluating the developmental status of fetal intestinal immune function. Figure 7 As shown, maternal supplementation with *Lactobacillus murineis* improves the polarization of intestinal macrophages in intrauterine growth-restricted fetal rats and inhibits the differentiation of quiescent macrophages into pro-inflammatory macrophages (CD11c). + See also Figure 7 A, B; P <0.05), but had no effect on the polarization of anti-inflammatory macrophages (CD206). + See also Figure 7 A, C; P >0.05); Simultaneously, maternal supplementation with *Lactobacillus murineis* significantly reduced markers of pro-inflammatory macrophages in the gut of intrauterine growth-restricted fetal rats. IRF5 Gene expression levels (see) Figure 7 D in P <0.05), while increasing pro-inflammatory macrophage markers IRF4 Gene expression levels (see) Figure 7 E in P <0.05); In addition, maternal supplementation with Lactobacillus murineis inhibited pro-inflammatory cytokines in the gut of intrauterine growth-restricted fetal rats. IL-1β and TNF-α Gene expression levels (see) Figure 7 F, G; P <0.05), and increased anti-inflammatory cytokine receptors. IL-4Ra Gene expression levels (see) Figure 7 H in P <0.05), while for anti-inflammatory cytokines IL-10 No significant effect on gene expression levels (see [link]). Figure 7 The I in P >0.05), thereby promoting the development of the immune function of fetal mice.

[0054] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.

Claims

1. Lactobacillus murineis ( Lactobacillus murinus Mafic1501, characterized in that, Deposited with China General Microbiological Culture Collection Center, and the deposit number is CGMCC NO.32163.

2. An inoculant characterized in that, Lactobacillus murinus according to claim 1 Lactobacillus murinus ) Mafic 1501.

3. An oral formulation for improving placental function in intrauterine growth restricted dams and / or development of intrauterine growth restricted fetuses, characterized in that, Lactobacillus murinus according to claim 1 Lactobacillus murinus Lactobacillus murinus ) Mafic 1501 or the bacterial agent according to claim 2.

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