Use of lactobacillus plantarum l168 in improving inflammation levels during pregnancy and in resisting drugs causing miscarriage due to excessive immune stimulation

Abstract

This invention relates to Lactobacillus plantarum ( Lactobacillus plantarum The application of L168 in improving inflammation levels during pregnancy and resisting abortion caused by excessive immune stimulation belongs to the field of microbial application technology. The *Lactobacillus plantarum* (…) Lactobacillus plantarum L168 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 13660. This invention relates to *Lactobacillus plantarum* (…). Lactobacillus plantarum) L168 effectively inhibits excessive inflammatory responses during pregnancy, reduces the risk of miscarriage caused by immune stimulation, improves the maternal immune microenvironment, and promotes healthy fetal development. As a natural probiotic preparation, it is highly safe and easy to take. It can be widely used in pregnancy health management products, especially in the adjunctive treatment and preventative intervention of high-risk pregnant women, and has significant clinical and commercial value.

Description

Technical Field

[0001] This invention relates to the application of Lactobacillus plantarum, specifically to the application of Lactobacillus plantarum L168 in drugs for improving inflammation levels during pregnancy and resisting abortion caused by excessive immune stimulation, belonging to the field of microbial application technology. Background Technology

[0002] Immune balance during pregnancy is crucial for maintaining a normal pregnancy. Excessive immune stimulation (such as excessive secretion of inflammatory factors) is a significant cause of miscarriage and adverse pregnancy outcomes. Excessive immune stimulation refers to the overactivation of the immune system, leading to an uncontrolled inflammatory response, which may trigger miscarriage or adverse pregnancy outcomes during pregnancy. Pregnancy outcome refers to the final result of the pregnancy process, including successful delivery or miscarriage. CCL2, also known as MCP-1, chemotactically attracts monocytes, T cells, and macrophages to sites of inflammation, potentially triggering local placental oxidative stress and tissue damage, and disrupting the maternal-fetal barrier. CXCL1 and CXCL2 chemotactically attract neutrophils, promoting acute inflammatory responses. After neutrophil aggregation, they release proteases (such as MMP-9) and reactive oxygen species (ROS), leading to placental vascular endothelial damage. CXCL10 activates Th1 / NK cells, promoting the secretion of pro-inflammatory cytokines (such as TNF-α and IFN-γ), exacerbating the inflammatory microenvironment. In recent years, the potential of probiotics in regulating immune function and anti-inflammation has attracted widespread attention. However, there are few studies on probiotic interventions for immune imbalance during pregnancy. Most existing products are general-purpose probiotic preparations, lacking specific strains that target pregnancy complications.

[0003] Lactobacillus plantarum is a common probiotic strain widely found in fermented foods. It can produce organic acids and bacteriocins with antibacterial properties through fermentation of various carbon sources, thus possessing antioxidant, gut-enhancing, and antibacterial preservation effects.

[0004] Patent document CN102533618A discloses a plant lactobacillus ( Lactobacillus plantarum CCFM8724 and its uses. This *Lactobacillus plantarum* CCFM8724 is extracted from fermented milk wine made by herders in Inner Mongolia and Xinjiang. It has the ability to inhibit the growth of *Campylobacter jejuni* in vitro, exhibits good acid and bile salt resistance, and has good adhesion to intestinal epithelial cells. It can inhibit the adhesion of *Campylobacter jejuni* to intestinal epithelial cells and inhibit the growth of *Campylobacter jejuni* in chicken meat.

[0005] Patent document CN 109234182A discloses a strain of lactic acid bacteria O2, which was isolated and screened from sourdough, a traditional leavening agent used in making steamed buns in northern China. This bacterium exhibits significant inhibitory effects against *Phytophthora capsici*, *Anthracis capsici*, *Escherichia coli*, *Salmonella*, *Shigella*, and *Cronobacter sakazakii*. Patent document CN 109234182A also discloses *Lactobacillus plantarum* (…).Lactobacillus plantarum ) ZJUFT34 can significantly improve the water content of mouse feces, and has the potential to relieve constipation; in addition, the plant lactobacillus has the ability to ferment milk and fermented soy milk.

[0006] The patent document CN 114404457B discloses the application of Lactobacillus plantarum L168 in preparing a medicine for delaying ovarian aging, which can increase the number of mature eggs of kdm5 gene deletion Drosophila and the hatching rate of offspring.

[0007] The existing probiotic products are mainly aimed at intestinal health or general immune enhancement, and lack special strains and application methods for excessive immune stimulation during pregnancy. For pregnancy inflammation, the existing technology has the following problems: first, it lacks specificity and cannot effectively intervene in pregnancy inflammation; second, the mechanism is not clear and the clinical effect is unstable. Therefore, in order to solve the problems of high miscarriage rate during pregnancy, limited intervention means for immune imbalance and excessive inflammation caused by excessive immune stimulation, it is urgent to obtain a probiotic strain for improving the level of inflammation during pregnancy and resisting miscarriage caused by excessive immune stimulation. SUMMARY

[0008] The present application provides a kind of Lactobacillus plantarum ( Lactobacillus plantarum L168 to solve the problems of high miscarriage rate during pregnancy, limited intervention means for immune imbalance and excessive inflammation caused by excessive immune stimulation.

[0009] The technical scheme adopted by the present application is as follows: a kind of Lactobacillus plantarum ( Lactobacillus plantarum L168 in the application of improving the level of inflammation during pregnancy and resisting miscarriage caused by excessive immune stimulation, the Lactobacillus plantarum ( Lactobacillus plantarum L168 strain was deposited with the China General Microbiological Culture Collection Center (CGMCC) on February 13, 2017, and the deposit address is No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road No. 1, and the deposit number is CGMCC No. 13660.

[0010] The Lactobacillus plantarum ( Lactobacillus plantarum L168 effectively inhibits excessive inflammatory response during pregnancy, reduces the risk of miscarriage caused by immune stimulation, improves the maternal immune microenvironment, promotes the healthy development of the fetus, and as a natural probiotic preparation, it is safe and easy to take. It can be widely used in pregnancy health management products, especially in the adjuvant therapy and preventive intervention of high-risk pregnant women, and has important clinical and commercial value.

[0011] Further, the medicine is composed of Lactobacillus plantarum ( Lactobacillus plantarum L168 agent and a pharmaceutically acceptable carrier.

[0012] Furthermore, the drug reduces the chemokines CCL2, CXCL1 / CXCL2 and CXCL10 in the placenta.

[0013] This invention relates to Lactobacillus plantarum ( Lactobacillus plantarum The application of strain L168 in controlling inflammation during pregnancy was scientifically verified to reduce miscarriage rate and improve pregnancy outcomes. In vitro experimental data showed that strain L168 reduced neutrophil aggregation by decreasing chemokines CCL2, CXCL1 / CXCL2, and CXCL10, thus regulating the maternal immune microenvironment and reducing the risk of miscarriage induced by excessive immune stimulation. The miscarriage rate in pregnant mice treated with strain L168 was 33.3% lower than the control group, and the expression levels of inflammatory chemokine genes were significantly decreased. Attached Figure Description

[0014] Figure 1 This is the animal experiment design process for an embodiment of the present invention;

[0015] Figure 2 The growth curve of Lactobacillus plantarum L168 of this invention;

[0016] Figure 3 The colony morphology of Lactobacillus plantarum L168 of this invention;

[0017] Figure 4 For the calculation and identification of the whole genome ANI of Lactobacillus plantarum L168 of the present invention;

[0018] Figure 5 This invention provides fetal mouse data after oral administration of live Lactobacillus plantarum L168 to pregnant mice;

[0019] Figure 6 This is data on abortion in pregnant mice after oral administration of live Lactobacillus plantarum L168 as described in this invention;

[0020] Figure 7 This invention provides data on the gene expression of live Lactobacillus plantarum L168 in the placenta of pregnant mice via gavage.

[0021] Figure 8 This is the result of an animal experiment involving the oral administration of live Lactobacillus plantarum L168, as described in this invention. Detailed Implementation

[0022] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] This invention relates to Lactobacillus plantarum ( Lactobacillus plantarum For the separation, screening, identification and preparation methods of L168, please refer to the patent document with application number 202111628233.2.

[0024] Example

[0025] In vitro experimental data showed that L168 strain reduced the aggregation of neutrophils by reducing chemotactic factors CCL2, CXCL1 / CXCL 2 and CXCL10, and reduced the level of inflammation.

[0026] Experimental method:

[0027] 1、As Figure 1 shown, 6-week-old mice (♂:♀=40:80) were purchased into an animal barrier facility, and after a week of adaptation, female mice (40) were given Lactobacillus plantarum L168 by gavage, with 1.5*10 9 CFU per mouse per day. The control group of female mice (40) was given phosphate buffered saline by gavage, and after 14 days of continuous gavage, mating was performed (when combining cages, male mice were first placed in the cage, preferably 2 days in advance, and then female mice were placed in the male mouse cage at a ratio of 1:2 or 1:1). The next day, the plug was checked, and each group of two mice that saw the plug was placed in a new cage box, and the E0.5 date was marked. The mice that did not see the plug were remated the next day.

[0028] 2、E12.5 intraperitoneal injection of Poly (I:C) (20 mg / kg).

[0029] 3、E14.5 cesarean section, take the pregnant mouse blood, do transcriptome; take the pregnant mouse uterus, embryo and placenta, take pictures and weigh.

[0030] 4、Statistical analysis of the abortion rate of this batch of experiments.

[0031] Collect placenta for transcriptome sequencing, count the expression of CCL2, CXCL1 / CXCL 2 and CXCL10, and count the abortion rate and fetal survival rate.

[0032] Data results: The levels of CCL2, CXCL1 / CXCL 2 and CXCL10 in the intervention group were significantly lower than those in the control group; the abortion rate decreased from 50% in the control group to 16.7%, and the fetal survival rate increased to 97.8%.

[0033] As Figure 2 shown, three Lactobacillus plantarum L168 monoclonal antibodies were isolated and labeled as L168-07, L168-08 and L168-09, inoculated in 96-well plates, 200ul of culture medium, cultured under anaerobic conditions for 24h, and the absorbance 600 (OD600) was detected every half hour, the data was recorded, and finally the growth curve of L168 was drawn. It was found that near 15h to the plateau phase, the absorbance 600 (OD600) value was the largest, indicating the largest number of viable bacteria.

[0034] As Figure 3As shown, Lactobacillus plantarum L168 was serially diluted and densely spread on MRS agar plates. After 48 hours of anaerobic culture, the colony morphology was observed by the naked eye. The L168 colonies had neat edges, were milky white, and appeared "bun-shaped". Some individual colonies were slightly wrinkled, which represented the morphology of Lactobacillus plantarum L168 at different growth stages. It was identified that the two morphologies of Lactobacillus plantarum L168 were identical in genome.

[0035] As shown in Figure 4, download Lactobacillus plantarum (Lactobacillus) from the Genebank database. Lactobacillus plantarum ) and Lactobacillus pentosaccharide ( Lactobacillus pentosus ) and Lactobacillus plantarum ( Lactobacillus plantarum L168-1 and Lactobacillus plantarum ( Lactobacillus plantarum The genome ANI of L168-7 was calculated, and species similarity was compared. Based on the ANI values, *Lactobacillus plantarum* (…) Lactobacillus plantarum L168-1 and Lactobacillus plantarum ( Lactobacillus plantarum L168-7 refers to the same bacterium, namely Lactobacillus plantarum. Lactobacillus plantarum L168, which is related to Lactobacillus plantarum ( Lactobacillus plantarum They are more closely related, with a genome similarity of over 99%, and are also related to *Lactobacillus pentosaceus* (…). Lactobacillus pentosus Despite being geographically distant, the similarity is approximately 80%, suggesting that *Lactobacillus plantarum* (…) Lactobacillus plantarum L168 belongs to Lactobacillus plantarum ( Lactobacillus plantarum ), rather than Lactobacillus pentosaceus ( Lactobacillus pentosus ).

[0036] As shown in Figure 5, this figure shows the gavage experiment of Lactobacillus plantarum L168 in mice. The results show that compared with the PBS+NS group, L168+NS, that is, gavage of Lactobacillus plantarum L168 in mice, did not affect the number of offspring. However, when pregnant mice were stimulated with Poly(I:C), Lactobacillus plantarum L168 significantly resisted the abortion response induced by Poly(I:C).

[0037] Note: Poly(I:C) acid (abbreviated as Poly(I:C), also known as polyinosinic acid-polycytidylic acid, is a polymer of inosinic acid and cytidine. It is an immune enhancer. Its sodium salt can be used to mimic viral infection.

[0038] Poly(I:C) interacts with Toll-like receptor (TLR)3, a protein on the surface of B cells, macrophages, and dendritic cells. Poly(I:C) is structurally similar to the double-stranded RNA of some viruses and can act as a natural enhancer of TLR3. Therefore, Poly(I:C) is often used as a double-stranded RNA analog in immunological studies.

[0039] As shown in Figure 6, the abortion rate of mice in both the PBS+NS group and the L168+NS group was 0, indicating that administering L168 plantarum via gavage alone without any stimulation would not have a negative effect on the mice. The abortion rate in the PBS+PIC group was 50%, while the abortion rate in the L168+PIC group was 16.7%, indicating that when pregnant mice were stimulated with Poly(I:C), gavage administration of L168 significantly resisted the excessive immune response induced by Poly(I:C) and reduced the probability of abortion.

[0040] As shown in Figure 7, transcriptomic gene expression was detected in the placentas of four groups of female mice. No difference in chemokine expression was found between the PBS+NS group (phosphate-buffered saline solution + saline) and the L168+NS group (Lactobacillus plantarum L168 + saline). When pregnant female mice were intraperitoneally injected with Poly(I:C), the chemokines CCL2, CCL2, CXCL1 / CXCL2, and CXCL10 were significantly increased in the placentas of the PBS+PIC group. However, in the L168+PIC group, due to L168 gavage intervention, the gene expression of chemokines CCL2, CXCL1 / CXCL2, and CXCL10 in the placentas of the female mice was significantly reduced. This indicates that gavage administration of Lactobacillus plantarum L168 reduced the immune stimulation induced by Poly(I:C), preventing miscarriage in female mice due to excessive immunization.

[0041] like Figure 8 As shown, the uterus and fetuses of four groups of female mice were photographed and counted. It was found that there was no change in the number of fetuses in the PBS+NC group (phosphate buffered saline solution + physiological saline) and the L168+NC group (Lactobacillus plantarum L168 + physiological saline). In the PBS+PIC group (phosphate buffered saline solution + Poly(I:C)), abortion occurred, and three fetuses were absorbed by the uterus due to abortion, leaving only the placenta. In the L168+PIC group (Lactobacillus plantarum L168 + Poly(I:C)), only one fetus aborted. The experiment significantly demonstrated that Lactobacillus plantarum L168 has a resistance effect against abortion caused by excessive immunity induced by Poly(I:C).

[0042] The above merely describes preferred embodiments of the present application, and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Lactobacillus plantarum ( Lactobacillus plantarum The use of L168 in medications to improve inflammation levels during pregnancy and combat miscarriage caused by excessive immune stimulation, wherein the *Lactobacillus plantarum* ( Lactobacillus plantarum Strain L168 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 13, 2017, with accession number CGMCCNo.13660.

2. Use according to claim 1, characterized in that, The medicament consists of Lactobacillus plantarum (L168) agent and a pharmaceutically acceptable carrier. Lactobacillus plantarum ) L168 agent and a pharmaceutically acceptable carrier.

3. Use according to claim 1, characterized in that, The drug reduces chemokines CCL2, CXCL1, CXCL2 and CXCL10 in the placenta.

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