Application of 3-oxoLCA in reducing oxidative stress and inflammation of pregnant maternal and offspring thereof

Abstract

The invention relates to medical and food use of bile acid derivatives. Specifically, the invention relates to an application of 3-oxoLCA in reducing oxidative stress and inflammation of a pregnant maternal and offspring of the pregnant maternal. Specifically, the invention relates to the use of 3-oxoLCA to reverse the influence of oxidative stress on the pregnant maternal, in particular, to maintain the average weight of a fetus, to significantly reduce the oxidative stress of the placenta and liver of the pregnant maternal and the inflammation or dysfunction of the placenta and liver caused thereby, and to improve the intestinal flora imbalance of the pregnant maternal. Further research finds that the 3-oxoLCA has long-term influence, and particularly, the 3-oxoLCA can obviously reduce the influence of oxidative stress in the gestation period on the oxidative stress and inflammation of offspring liver.

Description

TECHNICAL FIELD

[0001] The present application relates to the medical and food use of bile acid derivatives. Specifically, the present application relates to the application of 3-oxoLCA in reducing oxidative stress and inflammation of pregnant mothers and their offspring. BACKGROUND

[0002] In the late stage of pregnancy, maternal metabolism is enhanced to meet the energy and nutrient requirements of rapid fetal growth, but this process is accompanied by the production of excess reactive oxygen species (ROS), which in turn triggers maternal oxidative stress. This phenomenon is particularly pronounced in high-producing sows. When oxidative stress persists or is too strong, excess ROS will disrupt the intracellular redox balance, activate inflammatory signaling pathways such as nuclear factor-kappa B (NF-kappa B) and mitogen-activated protein kinase (MAPK), promote the release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), and ultimately induce or exacerbate systemic and local tissue (such as placenta and uterus) inflammation in the mother. Oxidative stress and inflammation also create an "oxidative stress-inflammation" positive feedback loop, where immune cell activation during the inflammation process further produces ROS, exacerbating oxidative stress damage. Together, they not only disrupt maternal energy balance and reduce reproductive performance, but also weaken piglet vitality, severely affecting the economic benefits of farming. More seriously, oxidative stress can further affect the placenta, inhibiting the proliferation and differentiation of trophoblast cells and limiting angiogenesis, thereby impairing the mother's nutrient transport to the fetus, ultimately affecting fetal metabolism and development. Studies in pigs have shown that low birth weight individuals are often associated with intestinal barrier dysfunction and systemic inflammatory response, manifested as inflammatory damage to the intestinal mucosa, intestinal flora imbalance, and immune homeostasis imbalance, which are closely related to significantly increased neonatal mortality.

[0003] 3-oxo-L-cholic acid (3-oxoLCA, 3-oxo-5β-cholanic acid-24-oic acid) is a natural bile acid metabolite produced by the oxidation of primary bile acids by intestinal flora 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH). Current research on 3-oxoLCA is limited, but its potential role in various physiological processes has gradually emerged. On the one hand, 3-oxoLCA can inhibit the differentiation of mouse intestinal Th17 cells by directly binding to the key transcription factor RORyt (retinoic acid-related orphan receptor gamma t), thereby regulating immune response; on the other hand, studies have shown that increasing 3-oxoLCA in neurons promotes ischemia-induced neuronal cell death, exhibiting a role in damaging nerve cells.

[0004] However, the prior art has not reported the effect of 3-oxoLCA on oxidative stress in the body. SUMMARY

[0005] Based on the prior art, the inventors unexpectedly found for the first time that 3-oxoLCA is able to reverse the effects of oxidative stress on the pregnant maternal, in particular, maintain the fetal equal weight, significantly reduce the oxidative stress of the placenta and liver of the pregnant maternal and the inflammation or dysfunction of the placenta and liver caused thereby, and improve the intestinal flora imbalance of the pregnant maternal. Further studies found that 3-oxoLCA has long-term effects, in particular, is able to significantly reduce the effects of oxidative stress of the pregnant maternal on the oxidative stress and inflammation of the offspring liver.

[0006] In this regard, the present application includes but is not limited to the following technical solutions: In one aspect, the present application provides the use of 3-oxoLCA in the preparation of a drug / composition for reducing the oxidative stress and / or inflammation of the pregnant maternal.

[0007] In one aspect, the oxidative stress of the pregnant maternal according to the present application is manifested as a decrease in the activity of antioxidant enzymes and an increase in the concentration of lipid peroxidation damage markers in the serum, preferably, the antioxidant enzyme is SOD, and / or the lipid peroxidation damage marker is MDA; and / or, the inflammation of the pregnant maternal is manifested as an increase in the level of inflammatory factors in the serum, preferably, the inflammatory factor is IL-6.

[0008] In one aspect, the oxidative stress of the pregnant maternal according to the present application is manifested as a decrease in the activity of SOD and an increase in the concentration of MDA in the serum.

[0009] In one aspect, the inflammation of the pregnant maternal according to the present application is manifested as an increase in the level of IL-6 in the serum.

[0010] In one aspect, the oxidative stress of the pregnant maternal according to the present application is manifested as a decrease in the activity of SOD and an increase in the concentration of MDA and the level of IL-6 in the serum.

[0011] In one aspect, the oxidative stress of the pregnant maternal according to the present application includes placental oxidative stress and / or liver oxidative stress; and / or, the inflammation of the pregnant maternal includes placental inflammation and / or liver inflammation.

[0012] In one aspect, the placental oxidative stress according to the present application is manifested as an increase in the expression of Keap1 and NQO1 genes and a decrease in the expression of Nrf2 and SOD1 genes in the placenta; and / or, the liver oxidative stress is manifested as an increase in the expression of Keap1 and NQO1 genes and a decrease in the expression of Nrf2, CAT and SOD1 genes in the liver; and / or, the placental inflammation is manifested as an increase in the expression of the inflammatory factor IL-6 gene in the placenta; and / or, the liver inflammation is manifested by an increase in the expression of the inflammatory factor IL-6 and / or IL-8 gene in the liver.

[0013] In one aspect, the present application also provides the use of 3-oxoLCA in the manufacture of a medicament / composition for reducing liver oxidative stress and / or inflammation of the offspring of a pregnant subject, wherein the pregnant subject has oxidative stress and / or inflammation.

[0014] In one aspect, the present application the liver oxidative stress of the offspring of a pregnant subject is manifested by a decrease in the expression of the SOD2 gene in the liver; and / or, the liver inflammation of the offspring of a pregnant subject is manifested by an increase in the expression of the inflammatory factor IL-8 gene in the liver.

[0015] In one aspect, the present application the pregnant subject has oxidative stress (e.g., including placental and / or liver oxidative stress) and / or inflammation (e.g., including placental and / or liver inflammation).

[0016] In one aspect, the present application the inflammation (e.g., including placental and / or liver inflammation of the pregnant subject and liver inflammation of the offspring of the pregnant subject) is caused by oxidative stress of the pregnant subject.

[0017] In yet another aspect, the present application provides the use of 3-oxoLCA in the manufacture of a medicament / composition for alleviating placental or liver dysfunction and / or improving bile acid metabolism or intestinal microecology, preferably, the placental or liver dysfunction and / or bile acid metabolism or intestinal microecology is the placental or liver dysfunction and / or bile acid metabolism or intestinal microecology of a pregnant subject.

[0018] In one aspect, the present application the placental dysfunction is manifested by a decrease in the expression of the placental function gene IGF2; and / or, the alleviating liver dysfunction is manifested by an increase in the expression of the bile acid receptor gene FXR and the rate-limiting enzyme gene CYP27A1 in the liver compared to the oxidative stress and / or inflammation state of the pregnant subject; and / or, the improving bile acid metabolism is manifested by an increase in the gene expression of FXR and CYP27A1 compared to the oxidative stress and / or inflammation state of the pregnant subject; and / or, the improving intestinal microecology is manifested by an increase in intestinal flora diversity, an increase in the abundance of beneficial bacteria, and an inhibition of abnormal bacteria overgrowth compared to the oxidative stress and / or inflammation state of the pregnant subject.

[0019] In one aspect, the present application the placental, liver dysfunction, bile acid metabolism abnormality, and / or intestinal microecology abnormality is caused by oxidative stress and / or inflammation of the pregnant subject.

[0020] In yet another aspect, the present application provides use of 3-oxoLCA in the manufacture of a medicament / composition for alleviating reduced fetal growth performance caused by oxidative stress and / or inflammation in a pregnant maternal, preferably, the reduced fetal growth performance is manifested as reduced litter weight and uniform weight as compared to the fetus produced by a maternal not suffering from oxidative stress and / or inflammation.

[0021] In one aspect, the pregnant maternal of the present application is a pregnant maternal of a human or an animal.

[0022] In one aspect, the pregnant maternal of the present application is a pregnant maternal of a human.

[0023] In one aspect, the animal of the present application is a mammal. In particular, the mammal is, for example, a mouse, a pig, a cow, a sheep, a horse, a camel, an alpaca, etc.

[0024] In one aspect, the mammal of the present application is a rodent; preferably, a mouse.

[0025] In one aspect, the oxidative stress, inflammation, placenta or liver dysfunction, reduced fetal growth performance, abnormal bile acid metabolism and / or abnormal intestinal microflora of the present application is induced by bisphenol A.

[0026] In one aspect, the oxidative stress of the pregnant maternal of the present application is induced by bisphenol A.

[0027] In one aspect, the oxidative stress of the present application is manifested as reduced activity of an antioxidant enzyme (e.g., SOD, SOD1 and / or SOD2).

[0028] In one aspect, the inflammation of the present application is manifested as increased expression of an inflammatory factor (e.g., IL-6 and / or IL-8).

[0029] In one aspect, the medicament or composition of the present application is orally administrable. In one aspect, the intake of 3-oxoLCA is preferably 10-50 mg / kg body weight per day, more preferably 20-40 mg / kg body weight per day, and most preferably 30 mg / kg body weight per day.

[0030] In one aspect, the composition of the present application, for example, a functional feed or a feed additive, can be further prepared as a pharmaceutically acceptable oral preparation, including but not limited to tablets, capsules, granules, oral liquid preparations (such as suspensions or solutions). In addition, the composition of the present application can also be added to food, beverage or dietary supplement as a nutritional supplement.

[0031] In one aspect, the expression of the genes involved in the present application can be detected according to the primer pairs of Table 1.

[0032] The beneficial effects of the present application include, but are not limited to, the following: The present application first discovers and verifies that 3-oxoLCA can alleviate the negative effects of oxidative stress during pregnancy on the mother and her offspring. Specifically, 3-oxoLCA can be safely used throughout pregnancy, reversing the decrease in fetal weight caused by maternal oxidative stress (e.g., induced by BPA) without affecting body weight and average daily feed intake during the experimental period, alleviating inflammation, placental nutrient transport disorders, and long-term damage to the offspring's liver and intestinal flora imbalance. More specifically, by supplementing 3-oxoLCA, the damage of oxidative stress is alleviated in the following five aspects: (1) Maternal antioxidant: regulating the Keap1 / Nrf2 pathway, reducing placental and liver oxidative damage; (2) Promote fetal development: up-regulate IGF2 and syncytinA gene expression, improve fetal weight; (3) Offspring mouse liver protection: up-regulate SOD2 expression, down-regulate IL-8 inflammatory factor; (4) Cross-generation flora regulation: indirectly improve the offspring's liver metabolic function through maternal flora changes; (5) Activation of bile acid receptor FXR, regulation of bile acid metabolism balance. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The experimental design for studying the effect of 3-oxoLCA on oxidative stress pregnant mice is shown.

[0034] Figure 2 The effect of 3-oxoLCA on serum-related indicators of oxidative stress pregnant mice is shown, wherein Figure A shows the results of maternal serum antioxidant indicators, and Figure B shows the results of maternal serum inflammatory indicators. * represents P <0.05; ** represents P <0.01.

[0035] Figure 3 The effect of 3-oxoLCA on placental antioxidant gene mRNA expression in oxidative stress pregnant mice is shown. * represents P <0.05; ** represents P <0.01.

[0036] Figure 4 The effect of 3-oxoLCA on placental inflammatory gene mRNA expression in oxidative stress pregnant mice is shown. * represents P <0.05.

[0037] Figure 5 The effect of 3-oxoLCA on placental function gene mRNA expression in oxidative stress pregnant mice is shown. * represents P <0.05.

[0038] Figure 6 Effect of 3-oxoLCA on mRNA expression of antioxidant genes in liver of oxidative stress pregnant dams. * represents P <0.05; ** represents P <0.01.

[0039] Figure 7 Effect of 3-oxoLCA on mRNA expression of inflammatory genes in liver of oxidative stress pregnant dams. * represents P <0.05; ** represents P <0.01.

[0040] Figure 8 Effect of 3-oxoLCA on mRNA expression of bile acid related genes in liver of oxidative stress pregnant dams. * represents P <0.05.

[0041] Figure 9 Effect of 3-oxoLCA on fecal microbiota diversity of oxidative stress pregnant dams, in which, A figure shows Simpson index analysis results, and B figure shows principal coordinate analysis results. * represents P <0.05; ** represents P <0.01.

[0042] Figure 10 Effect of 3-oxoLCA on fecal microbiota composition of oxidative stress pregnant dams, in which, A figure shows genus level composition results, and B figure shows genus level composition difference results.

[0043] Figure 11 Effect of 3-oxoLCA on fecal microbiota functional prediction of oxidative stress pregnant dams, in which, A figure shows functional abundance prediction results of 7a-hydroxysteroid dehydrogenase (7a-HSDH), and B figure shows bile acid: Na + co-transporter (BASS family) pathway functional prediction results.

[0044] Figure 12 Effect of 3-oxoLCA on mRNA expression of antioxidant genes in liver of offspring pups of oxidative stress pregnant dams. * represents P <0.05.

[0045] Figure 13 Effect of 3-oxoLCA on mRNA expression of inflammatory genes in liver of offspring pups of oxidative stress pregnant dams. * represents P <0.05; ** represents<0.01. P <0.01.

[0046] Figure 14The body weight and average daily food intake of the pregnant female mice during the experimental period were recorded. DETAILED DESCRIPTION

[0047] In order to make the technical personnel better understand the present application, the technical solutions of the present application will be described in detail below in combination with specific examples. It should be pointed out that the following detailed description is exemplary and only a part of the embodiments of the present application, but not all the embodiments.

[0048] Based on the examples in the present application, all other examples obtained by the ordinary skilled person in the art without making creative efforts shall belong to the scope of protection of the present application.

[0049] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary skilled person in the art to which the present application belongs. The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art, and can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to the conventional experimental methods or according to the operation instructions recommended by the suppliers.

[0050] Example 1 BPA induces oxidative stress in pregnant female mice and 3-oxoLCA treatment of oxidative stress in pregnant female mice 1. Test materials Animals: 30 SPF C57BL / 6 female mice (6-8 weeks old, body weight 20±2 g), 15 male mice; the mice were purchased from Sperof (Beijing) Biotechnology Co., Ltd; Reagents: bisphenol A (BPA) (purity > 98%), 3-oxoLCA (purity > 95%), corn oil (analytical pure); Instruments: automatic biochemical analyzer, fluorescence quantitative PCR instrument, 16S rRNA sequencing platform.

[0051] Bisphenol A (BPA, bisphenol A), also known as 2,2-bis (p-hydroxyphenyl) propane, is a widely used industrial compound, and its molecular formula is C 15 H 16O2. BPA is widely used in the manufacturing process of canned food and beverage packaging, baby bottles, water bottles, dental filling sealant, contact lenses, and hundreds of other daily necessities. Although BPA is a low-toxic substance, its potential harm to pregnant mothers and fetuses cannot be ignored. Studies have shown that BPA exposure can mimic female hormones, disrupt the endocrine system, and may cause reproductive problems, precocious puberty, affect breast development and reproductive function, and even cause hormone-related cancer. In addition, BPA can also cause maternal oxidative stress and affect the expression of UGT family genes (such as UGT1A1, UGT1A6, UGT1A9, and UGT2B1) in the placenta and fetal liver, leading to abnormal fetal development.

[0052] In the specific embodiments of the present application, pregnant female mice are treated with BPA to induce maternal oxidative stress to construct a model of oxidative stress during pregnancy.

[0053] 2. Experimental treatment and sample collection (1) Pairing and pregnancy confirmation Male and female mice were caged at a ratio of 1:2 overnight, and the next day the male and female mice were separated, and pregnancy was confirmed by checking the vaginal plug or sperm (GD1).

[0054] (2) Experimental grouping and treatment plan: Control (CON) group (n=10): corn oil (0.2 mL / each / d); Model (BPA) group (n=10): corn oil (0.2 mL / each / d) + BPA (200 mg / kg BW / d); Intervention (BPA+3-oxoLCA) group (n=10): corn oil (0.2 mL / each / d) + BPA (200 mg / kg BW / d) + 3-oxoLCA (30 mg / kg BW / d).

[0055] All pregnant female mice were fed with basic breeding feed. Corn oil, BPA and / or 3-oxoLCA were administered by oral gavage once a day, and the experimental period was 18.5 days of pregnancy, i.e. on the 18.5th day after pregnancy confirmation, the mice were sacrificed by cervical dislocation for sampling. See Figure 1 .

[0056] Example 2: Study of the effect of 3-oxoLCA on oxidative stress in pregnant female mice 1. Sample collection and analysis (1) Collection of maternal samples Maternal blood (carotid artery blood collection on the 18.5th day of pregnancy, serum separation), placental tissue, feces.

[0057] (2) Detection index Serum oxidative stress markers: The content of superoxide dismutase (SOD) (Cat No. A001-3-2) and malondialdehyde (MDA) (Cat No. A003-1-2) in serum samples were detected by commercial detection kit according to the instructions. The kit was purchased from Nanjing Jiancheng Bioengineering Institute, and the index determination method referred to the instructions of the kit.

[0058] Serum inflammatory factors: The levels of interleukin-6 (IL-6) (Cat No. YJ098430), interleukin-8 (IL-8) (Cat No. YJ063162), and interleukin-10 (IL-10) (Cat No. YJ037873) in serum were detected by enzyme-linked immunosorbent assay (ELISA) kit. The kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., and the index determination method referred to the instructions of the kit.

[0059] Gene expression: Total RNA was extracted from maternal placenta and liver samples using TRIzol reagent (Aidlab Biotechnologies Co., Ltd, Beijing, China) according to the manufacturer’s guidelines. RNA concentration and A260 / A280 ratio were determined using an Epoch microplate spectrophotometer (BioTek Instruments, Inc., VT, USA), and ratios between 2.0-2.2 were left for further analysis. Reverse transcription was performed using M5 Sprint qPCR RT kit and gDNA removal reagent (Mei5 Biotechnology, Co., Ltd, Beijing, China) according to the manufacturer’s protocol. Quantitative RT-PCR was performed using a QuantStudio™ RT-PCR system (Biorad) and SYBR Green reagent (Biorad, CA, USA). Primer sequences are shown in Table 1.

[0060] Table 1. RT-qPCR primer sequence list

[0061] Intestinal flora: Total microbial DNA was extracted from frozen fecal samples using a Fecal Genomic DNA Extraction Kit (Omega Bio-Tek, Norcross, GA, USA). DNA integrity was assessed by 1% agarose gel electrophoresis, and its concentration and purity were determined by absorbance at 260 nm and 280 nm (A260 / A280 ratio) using a NanoDrop™ One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA with an A260 / A280 ratio of 1.8-2.0 (high purity, no contaminants), intact bands (not degraded) in 1% agarose gel, and a concentration of ≥50 ng / μL was used as a template. The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified by PCR using specific primers 338F (5'-ACTCCTACGGGAGGCAGAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR product was purified (oxygen Biosciences, Union City, CA, USA), quantified, and used for library construction. Paired-end sequencing (2x250bp) was performed on the Illumina MiSeq platform. Raw sequencing data was denoised in QIIME2 (v2022.2) using DADA2 (version 1.26.0) and amplicon sequence variants (ASVs) were generated using default parameters. They were taxonomically annotated using the SILVA 138.2 reference database. Downstream analysis, sequences were first refined to the minimum number of sequences per sample. Subsequently, the Majorbio Cloud Platform (https: / / cloud.majorbio.com / ) was used for classification, community diversity (α / β-diversity), species difference (such as ANOSIM), and functional prediction (such as PICRUSt2) analysis. In addition, principal coordinate analysis (PCoA) based on bray-curtis, weighted UniFrac, and unweighted UniFrac distance was used to evaluate β diversity; Bile acid-related transport pathways: Based on 16S rRNA gene data, functional prediction was performed using PICRUSt2, and KEGG pathway annotation was used to infer the functional spectrum, i.e., 7α-HSDH, BASS family functional abundance prediction.

[0062] 2. Test results (1) Pregnancy of female rats Upon examination, all 30 female rats had sperm in their vaginas, in other words, all 30 female rats were pregnant.

[0063] (2) Statistical results of body weight and average daily feed intake of female rats during the test period Figure 14 The whole experimental period of the study on the maternal body weight (A) and average daily food intake (B) were systematically monitored. Statistical analysis showed that there was no significant difference between the BPA exposure group and the control group in body weight (P > 0.05) and daily food intake (P > 0.05). At the same time, the intervention group supplemented with 3-oxoLCA did not show significant difference with the basic group or the BPA exposure group in body weight dynamics and feeding behavior indicators (P > 0.05), suggesting that 3-oxoLCA had no obvious disturbance effect on the metabolic homeostasis and feeding regulation pathway of the mother rats within the experimental dose range. Figure 14 Figure 14 P P P

[0064] (3) Oxidative stress and inflammation in pregnant mothers Figure 2 The detection results of core antioxidant indicators and key inflammatory indicators in the serum of pregnant mothers are shown. Compared with the control group (CON), the serum of the bisphenol A exposure group (BPA) showed obvious imbalance characteristics of oxidative stress and inflammation. The superoxide dismutase (SOD) activity as the primary antioxidant enzyme in the body was significantly down-regulated, which indicated that BPA exposure weakened the ability of the mother rats to scavenge superoxide anion free radicals, directly breaking the oxidation-antioxidation balance. At the same time, the concentration of malondialdehyde (MDA), a marker of lipid peroxidation damage, was significantly up-regulated, indicating that free radical accumulation had caused oxidative damage to biological membranes, further proving the occurrence of oxidative stress, and the significant up-regulation of the pro-inflammatory cytokine interleukin-6 (IL-6) level reflected that BPA exposure not only induced oxidative stress but also activated the inflammatory response. When supplemented with 3-oxoLCA, the above abnormal changes caused by BPA were effectively reversed, which was specifically manifested as the significant increase of serum SOD activity compared with the BPA group (meaning that the antioxidant capacity of the mother rats was restored, and they could scavenge excess free radicals again), the synchronous decrease of MDA concentration (indicating that the degree of oxidative damage to biological membranes was reduced), and the significant decrease of IL-6 level (suggesting that the inflammatory response was effectively inhibited). In summary, this result clearly suggests that 3-oxoLCA supplementation can improve the oxidative stress disorder and inflammation imbalance in the serum of pregnant mothers caused by bisphenol A exposure in multiple dimensions by restoring antioxidant enzyme activity, reducing oxidative damage, and inhibiting the release of inflammatory factors.

[0065] (4) Expression of placental antioxidant, inflammation, and function-related genes in mother rats Figure 3 ​​​​​The mRNA expression results of placental antioxidant pathway key genes in pregnant dams are shown. Compared with the control group (CON), the gene expression of the bisphenol A exposure group (BPA) showed obvious abnormalities: the mRNA expression of Keap1 (negative regulator of the antioxidant pathway) and NQO1 (antioxidant compensatory gene) was significantly up-regulated, while the mRNA expression of Nrf2 (core transcription factor of the antioxidant pathway) and SOD1 (key intracellular antioxidant enzyme gene) was significantly down-regulated. This change indicates that BPA exposure weakens the activation ability of the placental antioxidant pathway by enhancing the inhibition of Keap1 on Nrf2, leading to a decrease in the synthesis of core antioxidant enzymes; while the up-regulation of NQO1 is a passive compensatory response of the placenta to oxidative damage, but cannot reverse the downward trend of the overall antioxidant capacity, directly reflecting the oxidative stress state of the placenta. Supplementing 3-oxoLCA can reverse some of the adverse effects of BPA, specifically by significantly down-regulating Keap1 mRNA expression and significantly up-regulating SOD1 mRNA expression, suggesting that it can restore the placental antioxidant defense function by relieving the inhibition of the Nrf2 pathway and promoting the synthesis of antioxidant enzymes.

[0066] Figure 4 The mRNA expression results of placental inflammatory genes in the BPA group show that the expression of the pro-inflammatory cytokine IL-6 is significantly up-regulated. Combined with the oxidative stress caused by the down-regulation of SOD1 in the CON group, Figure 3 BPA exposure-induced placental oxidative damage further activates the placental inflammatory response; while the 3-oxoLCA supplement group shows a down-regulation trend of IL-6, indicating that 3-oxoLCA has a potential inhibitory effect on placental local inflammation while restoring antioxidant capacity.

[0067] Figure 5 The mRNA expression results of placental functional genes in the BPA group show that compared with the control group, the expression of IGF2 (a key gene that regulates placental nutrient transport and angiogenesis) is significantly down-regulated, suggesting that BPA exposure damages the placental nutrient supply capacity; while supplementing 3-oxoLCA not only reverses the inhibitory effect of BPA on IGF2, but also significantly up-regulates the expression of syncytinA (a core gene that mediates the formation of placental syncytiotrophoblasts and maintains the function of the maternal-fetal barrier). This indicates that 3-oxoLCA can improve functional gene expression, not only repair the placental nutrient transport efficiency, but also maintain its structural integrity, providing protection for maternal-fetal material exchange and pregnancy stability.

[0068] (5) Gene expression related to antioxidant, inflammation, and bile acid metabolism in the livers of pregnant dams Figure 6The mRNA expression results of key genes of the antioxidant pathway in the liver of pregnant dams are shown. Compared with the control group (CON), the gene expression of the bisphenol A exposure group (BPA) was significantly abnormal: the mRNA expression of Keap1 (negative regulator of the antioxidant pathway) and NQO1 (antioxidant compensatory gene) was significantly up-regulated, while the mRNA expression of Nrf2 (core transcription factor of the antioxidant pathway), CAT (hydrogen peroxide enzyme gene, clearing hydrogen peroxide) and SOD1 (superoxide dismutase gene, clearing superoxide anion) was significantly down-regulated. This indicates that BPA exposure blocks the activation of the liver antioxidant pathway by enhancing the inhibition of Keap1 on Nrf2, resulting in reduced synthesis of core antioxidant enzymes; while the up-regulation of NQO1 is a passive compensation of the liver in response to oxidative damage, which cannot make up for the decline in overall antioxidant capacity, directly reflecting that the liver is in an oxidative stress state. Supplementing 3-oxoLCA can reverse some of the adverse effects of BPA, not only significantly down-regulating the mRNA expression of Keap1, significantly up-regulating the mRNA expression of SOD1, but also showing an up-regulation trend of Nrf2 mRNA expression, suggesting that it can restore the liver antioxidant defense function by relieving the inhibition of Nrf2 pathway and promoting the synthesis of antioxidant enzymes.

[0069] Figure 7 The mRNA expression results of liver inflammation genes of the bisphenol A exposure group (BPA) show that, compared with the control group (CON), the mRNA expression of pro-inflammatory factors IL-6 (mediating the inflammatory cascade) and IL-8 (recruiting inflammatory cells) is significantly up-regulated. Combined with the mRNA expression results of key genes of the antioxidant pathway in the liver of pregnant dams, Figure 6 the occurrence of oxidative stress in the liver, BPA-induced liver oxidative damage further activates inflammation; while supplementing 3-oxoLCA can significantly down-regulate the mRNA expression of IL-6, indicating that it can effectively inhibit local inflammatory response and reduce inflammatory damage while relieving liver oxidative stress.

[0070] Figure 8 The mRNA expression results of liver bile acid metabolism-related genes of the bisphenol A exposure group (BPA) show that, compared with the control group (CON) and the bisphenol A exposure group (BPA), supplementing 3-oxoLCA can significantly up-regulate the mRNA expression of FXR (bile acid nuclear receptor, regulating bile acid synthesis and transport) and CYP27A1 (rate-limiting enzyme of bile acid synthesis, catalyzing the conversion of cholesterol to bile acid). This suggests that 3-oxoLCA can regulate the synthesis and metabolic balance of liver bile acid by activating the FXR signaling pathway and promoting the expression of CYP27A1, and this action may be one of the important mechanisms for its improvement of liver oxidative stress and inflammation.

[0071] (6) Intestinal flora of feces of dams The flora structure in the intestinal contents was analyzed by 16S rRNA sequencing, and the specific analysis indicators and results are described as follows: At the level of flora diversity, Figure 9Analysis of α-diversity (Simpson index) in gut microbiota showed that 3-oxoLCA supplementation significantly reduced the Simpson index compared to the bisphenol A (BPA) exposure group. Since a lower Simpson index indicates higher species richness and evenness of the gut microbiota, this result suggests that 3-oxoLCA can improve the abnormal α-diversity of the gut microbiota caused by BPA exposure and restore the balance of the internal structure of the microbiota. Figure 9 The results of β-diversity analysis (PCoA) of B showed that the gut microbiota samples from the 3-oxoLCA supplementation group and the BPA group were significantly separated along the principal component axis, indicating that 3-oxoLCA can significantly change the overall community composition structure of gut microbiota under BPA exposure and promote the transformation of the microbiota structure towards a more normal state.

[0072] At the level of fungal composition Figure 10 The results showed that the abundance of bacterial genera was significantly abnormal in the bisphenol A exposure group compared with the CON group: Dubosiella, Bifidobacterium, Bacillus, Muribaculum The abundance of [unclear] was significantly upregulated, while norank_f__Muribaculaceae, Odoribacter, Alistipes, Rikenella The abundance of these bacteria was significantly reduced. Supplementation with 3-oxoLCA specifically modulated the abnormal changes in some of these bacterial genera, specifically by significantly increasing the levels of the previously inhibited β-oxoLCA in the bisphenol A-exposed group. Alistipes, Rikenella The abundance of (beneficial bacteria known to participate in intestinal bile acid conversion and anti-inflammatory processes) was significantly reduced, while the abnormally elevated levels in the bisphenol A exposure group were also significantly decreased. Bifidobacterium and normal abundance Lactobacillus Abundance. These results indicate that 3-oxoLCA supplementation reconstructs the genus-level composition of the gut microbiota by targeting and restoring the abundance of beneficial bacteria and inhibiting the excessive proliferation of abnormal bacteria. Moreover, its regulatory effect has obvious genus-specificity, providing direct evidence for improving the intestinal microecological imbalance caused by bisphenol A exposure.

[0073] (7) Bile acid metabolism pathway in fecal microorganisms of female rats See Figure 11 Based on gut microbiota functional prediction analysis, the functional abundance of key pathways in bile acid metabolism in the feces of pregnant female mice showed inter-group differences. Compared with the control group (CON), the bisphenol A exposure group and the 3-oxoLCA supplementation group showed common changes, with a significant upregulation of the functional abundance of 7α-hydroxysteroid dehydrogenase (7α-HSDH). Figure 11 A). 7α-HSDH is a core enzyme in the microbial-mediated conversion of primary bile acids to secondary bile acids. Its upregulation indicates that both BPA exposure and 3-oxoLCA supplementation can alter the conversion activity of intestinal bile acids, thereby affecting the balance of primary / secondary bile acid ratios in the body and indirectly related to liver oxidative stress and metabolic function.

[0074] The difference between the two groups mainly reflects in the bile acid transport pathway: the bisphenol A exposure group significantly down-regulates the bile acid: Na + Co-transporter (BASS family) pathway function Figure 11 B). BASS family is the core carrier of intestinal absorption and transport of bile acids, and the down-regulation of its pathway function will directly inhibit the enterohepatic circulation of bile acids, leading to bile acid absorption disorder and increased liver synthesis burden, and even indirectly exacerbating liver oxidative-inflammation damage; while the 3-oxoLCA supplement group did not show significant effects on this transport pathway, suggesting that 3-oxoLCA can maintain normal bile acid transport function by avoiding the inhibition of BASS family pathway by bisphenol A exposure, providing a basis for repairing bile acid metabolism disorder and protecting liver function.

[0075] Example 3: Study of the effect of 3-oxoLCA on oxidative stress maternal offspring (1) Fetal weight On the 18.5th day of pregnancy, the mother rats were euthanized according to the animal ethics standards, and the dissection operation was carried out after confirming the absence of vital signs. The mother rats were fixed on the dissection table with the abdomen facing up, the abdominal skin was disinfected with 75% alcohol, the skin and abdominal muscle were cut along the midline to expose the uterine horn, the uterus and surrounding connective tissue were carefully separated and completely removed, and placed in sterile saline. Then use tweezers to gently tear the uterine wall and remove the fetuses one by one, remove the fetal membranes and umbilical cords and place them in new sterile saline for rinsing to remove residual blood and tissue debris. Finally, the fetal weight was measured: the electronic balance was adjusted to 0.001 g and calibrated, the surface moisture of each fetus was uniformly absorbed with sterile filter paper, and placed quickly in the center of the weighing pan. After the reading was stable, the weight was recorded, and the operation was repeated to complete the weighing of all fetuses, and the number of fetuses was recorded simultaneously, and finally the average weight of the litter was calculated for subsequent difference analysis between groups.

[0076] As shown in Table 2, compared with the control group (CON), the fetal litter weight (reflecting the overall growth level of the litter) and the fetal average weight (reflecting the average growth status of individual fetuses) of the bisphenol A exposure group were significantly reduced, indicating that BPA exposure had a significant inhibitory effect on fetal intrauterine growth, leading to reduced fetal growth performance. The supplementation of 3-oxoLCA can effectively improve this situation, with a significant increase in fetal average weight, suggesting that 3-oxoLCA supplementation can specifically alleviate the inhibitory effect of BPA exposure on fetal growth, to some extent reversing the problem of decreased fetal average weight caused by BPA, providing experimental evidence for improving the intrauterine growth environment of fetuses.

[0077] Table 2: Effect of 3-oxoLCA on the reproductive performance of BPA-induced pregnant rats

[0078] Note: a, b, c different superscripts indicate significant differencesP <0.05). IUGR: intrauterine growth restriction.

[0079] (2) Offspring mice liver antioxidant and inflammation related gene expression The offspring mice liver samples were collected and the mRNA expression of antioxidant and inflammation related genes were analyzed according to the detection method in Example 1. The results showed that BPA exposure had significant transgenerational effects on the liver gene expression of offspring mice. Compared with the control group (CON), the mRNA expression of antioxidant gene SOD2 (mitochondrial superoxide dismutase, responsible for scavenging free radicals from mitochondria) was significantly down-regulated in the BPA exposure group, indicating that BPA exposure weakened the antioxidant defense capacity of the liver mitochondria of offspring mice, increasing the risk of oxidative stress in the liver; while the supplementation of 3-oxoLCA could effectively reverse this abnormality, significantly up-regulating the mRNA expression of SOD2 in the liver of offspring mice, suggesting that it could enhance the ability of offspring mice to cope with oxidative damage by restoring the expression of mitochondrial antioxidant enzyme genes (see Figure 12 ).

[0080] Figure 13 It was shown that BPA exposure could significantly increase the gene expression level of pro-inflammatory factor IL-8 in the liver of offspring mice (P < 0.05), while the supplementation of 3-oxoLCA in the diet of the mother mice significantly reduced this index to near the level of the control group (P < 0.05). IL-8 is a key signaling molecule in the inflammatory response, and its expression is usually elevated in tissue damage and immune abnormalities. The above results showed that 3-oxoLCA could effectively inhibit BPA-induced liver inflammation and had potential anti-inflammatory protective effects. P <0.05), while the supplementation of 3-oxoLCA in the diet of the mother mice significantly reduced this index to near the level of the control group (P < 0.05). IL-8 is a key signaling molecule in the inflammatory response, and its expression is usually elevated in tissue damage and immune abnormalities. The above results showed that 3-oxoLCA could effectively inhibit BPA-induced liver inflammation and had potential anti-inflammatory protective effects. P ​

Claims

1. Use of 3-oxoLCA in the preparation of pharmaceuticals / compositions for reducing maternal oxidative stress and / or inflammation during pregnancy.

2. The use according to claim 1, characterized in that, The oxidative stress in pregnant women is manifested by decreased serum antioxidant enzyme activity and increased concentrations of lipid peroxidation damage markers. And / or, the inflammation in the pregnant mother is manifested by an increase in the level of inflammatory factors in the serum.

3. The use according to claim 1, characterized in that, The maternal oxidative stress during pregnancy includes placental oxidative stress and / or hepatic oxidative stress; And / or, the maternal inflammation during pregnancy includes placental inflammation and / or liver inflammation.

4. The use according to claim 3, characterized in that, The placental oxidative stress was manifested by increased expression of Keap1 and NQO1 genes and decreased expression of Nrf2 and SOD1 genes in the placenta. And / or, the liver oxidative stress is manifested as increased expression of Keap1 and NQO1 genes and decreased expression of Nrf2, CAT and SOD1 genes in the liver; And / or, the placental inflammation is characterized by increased expression of the inflammatory factor IL-6 gene in the placenta; And / or, the liver inflammation is characterized by increased expression of the genes for the inflammatory factors IL-6 and / or IL-8 in the liver.

5. Use of 3-oxoLCA in the preparation of a medicament / composition for reducing hepatic oxidative stress and / or inflammation in offspring of a pregnant mother, wherein the pregnant mother is experiencing oxidative stress and / or inflammation.

6. The use according to claim 5, characterized in that, The oxidative stress in the offspring of the mothers during pregnancy was manifested by decreased expression of the SOD2 gene in the liver; And / or, liver inflammation in the offspring of the pregnant mother is manifested by increased expression of the inflammatory cytokine IL-8 gene in the liver.

7. Use of 3-oxoLCA in the preparation of a medicament / composition for relieving placental or liver dysfunction and / or improving bile acid metabolism or gut microbiota, wherein the placental or liver dysfunction and / or bile acid metabolism or gut microbiota is a maternal placental or liver dysfunction and / or bile acid metabolism or gut microbiota during pregnancy.

8. The use according to claim 7, characterized in that, Placental dysfunction is characterized by decreased expression of the placental function gene IGF2; And / or, relief of liver dysfunction is manifested by increased expression of the bile acid receptor gene FXR and the rate-limiting enzyme gene CYP27A1 in the liver compared to maternal oxidative stress and / or inflammatory state during pregnancy; And / or, the improvement in bile acid metabolism is manifested by increased expression of the FXR and CYP27A1 genes compared to maternal oxidative stress and / or inflammatory status during pregnancy; And / or, the improvement in gut microbiota is manifested by increasing gut microbiota diversity, increasing the abundance of beneficial bacteria, and inhibiting the excessive proliferation of abnormal bacteria compared to the oxidative stress and / or inflammatory state of the mother during pregnancy.

9. Use of 3-oxoLCA in the preparation of a medicament / composition for alleviating reduced fetal growth performance caused by maternal oxidative stress and / or inflammation during pregnancy.

10. The use according to any one of claims 1-9, characterized in that, The pregnant mother refers to a pregnant human or animal mother.

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