3-oxoLCA for reducing oxidative stress and inflammation in pregnant dams and their offspring

Abstract

The present invention relates to the medical and food use of bile acid derivatives. Specifically, the present invention relates to the use of 3-oxoLCA in reducing oxidative stress and inflammation in pregnant mothers and their offspring. Specifically, the present invention relates to the use of 3-oxoLCA to reverse the effects of oxidative stress on pregnant mothers, in particular, to maintain fetal equal weight, significantly reduce placental and liver oxidative stress in pregnant mothers and the resulting inflammation or dysfunction of the placenta and liver, and to improve the intestinal flora imbalance of pregnant mothers. Further studies have found that 3-oxoLCA has long-term effects, in particular, can significantly reduce the effects of oxidative stress during pregnancy on oxidative stress and inflammation in the liver of offspring.

Description

Technical Field

[0001] This invention relates to the medical and food uses of bile acid derivatives. Specifically, this invention relates to the use of 3-oxoLCA in reducing oxidative stress and inflammation in pregnant women and their offspring. Background Technology

[0002] In late pregnancy, maternal metabolism increases to meet the energy and nutrient demands of the rapidly growing fetus. However, this process is accompanied by the excessive production of reactive oxygen species (ROS), leading to maternal oxidative stress. This phenomenon is particularly pronounced in high-producing sows. When oxidative stress is persistent or excessively high, excess ROS disrupts intracellular redox balance, activates inflammatory signaling pathways such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK), and promotes the release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), ultimately inducing or exacerbating systemic and local inflammatory responses in the mother (such as in the placenta and uterus). Furthermore, oxidative stress and inflammation create a positive feedback loop of "oxidative stress-inflammation." During the inflammatory response, immune cell activation further generates ROS, exacerbating oxidative stress damage. The combined effect of these two factors not only disrupts maternal energy balance and reduces reproductive performance but also weakens piglet vitality, severely impacting the economic benefits of pig farming. More seriously, oxidative stress can further affect the placenta, inhibiting trophoblast cell proliferation and differentiation, and limiting angiogenesis, thereby weakening the maternal nutrient delivery to the fetus and ultimately affecting fetal metabolism and development. Studies using pigs as an example have shown that low birth weight individuals are often accompanied by intestinal barrier dysfunction and systemic inflammatory responses, manifested as inflammatory damage to the intestinal mucosa, intestinal flora imbalance, and immune homeostasis imbalance. These pathological changes are closely related to a significantly increased neonatal mortality rate.

[0003] 3-oxo-L-cholic acid (3-oxoLCA, 3-oxo-5β-cholan-24-acid) is a natural bile acid metabolite produced by the oxidation of primary bile acids by intestinal flora 7α-hydroxysteroid dehydrogenase (7α-HSDH). Current research on 3-oxoLCA is relatively limited, but its potential roles in various physiological processes are gradually becoming apparent. On the one hand, 3-oxoLCA can regulate immune responses by directly binding to the key transcription factor RORγt (retinoic acid-associated orphan receptor γt), inhibiting the differentiation of Th17 cells in the mouse gut. On the other hand, studies have shown that increasing intraneuronal 3-oxoLCA promotes ischemia-induced neuronal cell death, exhibiting a neuronal damaging effect.

[0004] However, existing technologies have not reported the effects of 3-oxoLCA on oxidative stress in the body. Summary of the Invention

[0005] Building upon existing technologies, the inventors unexpectedly discovered for the first time that 3-oxoLCA can reverse the effects of oxidative stress on pregnant women, particularly maintaining fetal average weight, significantly reducing oxidative stress in the placenta and liver during pregnancy and the resulting inflammation or dysfunction, as well as improving maternal gut microbiota imbalance. Further research revealed that 3-oxoLCA has long-term effects, particularly significantly reducing the impact of oxidative stress during pregnancy on offspring liver oxidative stress and inflammation.

[0006] In this regard, the present invention includes, but is not limited to, the following technical solutions:

[0007] In one aspect, the present invention provides the use of 3-oxoLCA in the preparation of a medicament / composition for reducing oxidative stress and / or inflammation in pregnant women.

[0008] In one aspect, the oxidative stress of the pregnant mother described in this invention is manifested as a decrease in the activity of antioxidant enzymes in serum and an increase in the concentration of lipid peroxidation damage markers. Preferably, the antioxidant enzyme is SOD, and / or the lipid peroxidation damage marker is MDA.

[0009] And / or, the inflammation in the pregnant mother is manifested by an increase in the level of inflammatory factors in the serum, preferably, the inflammatory factor is IL-6.

[0010] In one aspect, the oxidative stress of pregnant mothers described in this invention is manifested as decreased SOD activity and increased MDA concentration in serum.

[0011] In one aspect, the inflammatory manifestation of the pregnant mother described in this invention is an increase in serum IL-6 levels.

[0012] In one aspect, the oxidative stress of pregnant mothers described in this invention is manifested as decreased SOD activity and increased MDA concentration and IL-6 levels in serum.

[0013] In one aspect, the oxidative stress of the pregnant mother according to the present invention includes placental oxidative stress and / or liver oxidative stress; and / or, the inflammation of the pregnant mother includes placental inflammation and / or liver inflammation.

[0014] In one aspect, the placental oxidative stress described in this invention is manifested as increased expression of Keap1 and NQO1 genes and decreased expression of Nrf2 and SOD1 genes in the placenta.

[0015] 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;

[0016] And / or, the placental inflammation is characterized by increased expression of the inflammatory factor IL-6 gene in the placenta;

[0017] 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.

[0018] In one aspect, the present invention also provides the use of 3-oxoLCA in the preparation of a medicament / composition for reducing hepatic oxidative stress and / or inflammation in offspring of pregnant mothers, said pregnant mothers having oxidative stress and / or inflammation.

[0019] In one aspect, the oxidative stress in the offspring of the pregnant mothers described in this invention is manifested by decreased expression of the SOD2 gene in the liver; and / or, the liver inflammation in the offspring of the pregnant mothers is manifested by increased expression of the inflammatory factor IL-8 gene in the liver.

[0020] In one aspect, the pregnant mother described in this invention experiences oxidative stress (e.g., including placental and / or liver oxidative stress) and / or inflammation (e.g., including placental and / or liver inflammation).

[0021] In one aspect, the inflammation described in this invention (e.g., placental and / or liver inflammation in pregnant mothers and liver inflammation in the offspring of pregnant mothers) is caused by oxidative stress in pregnant mothers.

[0022] In another aspect, the present invention provides the use of 3-oxoLCA in the preparation of a medicament / composition for alleviating placental or liver dysfunction and / or improving bile acid metabolism or gut microbiota, preferably wherein the placental or liver dysfunction and / or bile acid metabolism or gut microbiota is a placental or liver dysfunction and / or bile acid metabolism or gut microbiota of a pregnant mother.

[0023] In one aspect, the placental dysfunction described in this invention is manifested as decreased expression of the placental functional gene IGF2; and / or, the relief of liver dysfunction is manifested as increased 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 inflammatory state of the pregnant mother; and / or, the improvement of bile acid metabolism is manifested as increased gene expression of FXR and CYP27A1 compared to the oxidative stress and / or inflammatory state of the pregnant mother; and / or, the improvement of gut microbiota is manifested as increased gut microbiota diversity, increased abundance of beneficial bacteria, and inhibition of excessive proliferation of abnormal bacteria compared to the oxidative stress and / or inflammatory state of the pregnant mother.

[0024] In one aspect, the placenta, liver dysfunction, bile acid metabolism abnormalities, and / or intestinal microecological abnormalities described in this invention are caused by maternal oxidative stress and / or inflammation during pregnancy.

[0025] In another aspect, the present invention provides the 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, preferably, the reduced fetal growth performance is manifested as reduced litter weight and average weight compared to fetuses born to mothers without oxidative stress and / or inflammation.

[0026] In one aspect, the pregnant mother described in this invention is a pregnant mother of a human or animal.

[0027] In one aspect, the pregnant mother described in this invention is a human pregnant mother.

[0028] In one aspect, the animal described in this invention is a mammal. In particular, mammals include, for example, rats, pigs, cattle, sheep, horses, camels, alpacas, etc.

[0029] In one aspect, the mammal described in this invention is a rodent; preferably, it is a mouse.

[0030] In one aspect, the oxidative stress, inflammation, placental or liver dysfunction, reduced fetal growth performance, bile acid metabolism abnormalities, and / or gut microbiota abnormalities described in this invention are induced by bisphenol A.

[0031] In one aspect, the oxidative stress in pregnant mothers described in this invention is induced by bisphenol A.

[0032] In one aspect, the oxidative stress described in this invention is manifested as a decrease in the activity of antioxidant enzymes (e.g., SOD, SOD1 and / or SOD2).

[0033] In one aspect, the inflammation described in this invention is characterized by increased expression of inflammatory factors (e.g., IL-6 and / or IL-8).

[0034] In one aspect, the drug or composition of the present invention is orally edible. 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.

[0035] In one aspect, the compositions of the present invention may, for example, be functional feeds or feed additives, and may be further prepared into pharmaceutically acceptable oral formulations, including but not limited to tablets, capsules, granules, and oral liquid formulations (such as suspensions or solutions). Furthermore, the compositions of the present invention may also be added to foods, beverages, or dietary supplements as nutritional supplements.

[0036] In one aspect, the expression of the genes involved in this invention can be detected using the primer pairs in Table 1.

[0037] The beneficial effects of the present invention include, but are not limited to, the following:

[0038] This invention is the first to discover and verify that 3-oxoLCA can alleviate the negative effects of oxidative stress during pregnancy on the mother and offspring. Specifically, 3-oxoLCA can be safely used throughout pregnancy, reversing the decrease in fetal average weight caused by maternal oxidative stress (e.g., induced by BPA) without affecting the offspring's weight or average daily food intake during the experimental period, and alleviating inflammation, placental nutrient transport disorders, long-term liver damage in offspring, and gut microbiota imbalance. More specifically, by supplementing with 3-oxoLCA, the damage caused by oxidative stress is alleviated in the following five ways:

[0039] (1) Maternal antioxidant: Regulates the Keap1 / Nrf2 pathway to reduce placental and liver oxidative damage;

[0040] (2) Promotes fetal development: Upregulates the expression of IGF2 and syncytinA genes, and improves fetal weight;

[0041] (3) Liver protection in offspring mice: upregulation of SOD2 expression and downregulation of IL-8 inflammatory factor;

[0042] (4) Transgenerational microbiota regulation: indirectly improves the liver metabolic function of offspring through changes in maternal microbiota;

[0043] (5) Activate bile acid receptor FXR and regulate bile acid metabolism balance. Attached Figure Description

[0044] Figure 1 This study demonstrates the experimental design for investigating the effects of 3-oxoLCA on pregnant rats under oxidative stress.

[0045] Figure 2 This diagram illustrates the effects of 3-oxoLCA on serum-related parameters in pregnant mice under oxidative stress. Figure A shows the results of serum antioxidant parameters in maternal mice, and Figure B shows the results of serum inflammatory parameters in maternal mice. * indicates... P <0.05; ** represents P <0.01.

[0046] Figure 3 This shows the effect of 3-oxoLCA on the mRNA expression of antioxidant genes in the placenta of pregnant mice under oxidative stress. * indicates P <0.05; ** represents P <0.01.

[0047] Figure 4 This shows the effect of 3-oxoLCA on the mRNA expression of placental inflammation genes in pregnant mice under oxidative stress. * indicates P <0.05.

[0048] Figure 5 This shows the effect of 3-oxoLCA on the mRNA expression of placental functional genes in pregnant mice under oxidative stress. * indicates P <0.05.

[0049] Figure 6 This shows the effect of 3-oxoLCA on the expression of antioxidant gene mRNA in the liver of pregnant mice under oxidative stress. * indicates P <0.05; ** represents P <0.01.

[0050] Figure 7 This shows the effect of 3-oxoLCA on the expression of liver inflammation genes mRNA in pregnant mice under oxidative stress. * indicates P <0.05; ** represents P <0.01.

[0051] Figure 8 This shows the effect of 3-oxoLCA on the expression of bile acid-related gene mRNA in the liver of pregnant mice under oxidative stress. * indicates P <0.05.

[0052] Figure 9 This diagram illustrates the effect of 3-oxoLCA on the fecal microbial community diversity of pregnant mice under oxidative stress. Figure A shows the results of Simpson index analysis, and Figure B shows the results of principal coordinate analysis. * indicates... P <0.05; ** represents P <0.01.

[0053] Figure 10 The effect of 3-oxoLCA on the fecal microbial community composition of pregnant mice under oxidative stress is shown in Figure A, which shows the genus-level composition results, and Figure B shows the genus-level composition difference results.

[0054] Figure 11 This study demonstrates the effect of 3-oxoLCA on the functional prediction of fecal microbiota in pregnant mice under oxidative stress. Figure A shows the predicted functional abundance of 7α-hydroxysteroid dehydrogenase (7α-HSDH), and Figure B shows the bile acid:Na + Predicted results of the function of the cotransporter (BASS family) pathway.

[0055] Figure 12 This shows the effect of 3-oxoLCA on the expression of antioxidant gene mRNA in the liver of offspring mice from pregnant mothers under oxidative stress. * indicates P <0.05.

[0056] Figure 13 This shows the effect of 3-oxoLCA on the expression of hepatic inflammatory gene mRNA in offspring of pregnant mice under oxidative stress. * indicatesP <0.05; ** represents P <0.01.

[0057] Figure 14 This shows the weight and average daily food intake of pregnant female mice during the experimental period. Detailed Implementation

[0058] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0059] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0060] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0061] Example 1: BPA-induced oxidative stress in pregnant rats and 3-oxoLCA treatment of oxidative stress in pregnant rats

[0062] 1. Experimental materials

[0063] Animals: 30 female SPF-grade C57BL / 6 mice (6-8 weeks old, weighing 20±2 g) and 15 male mice; mice were purchased from SPF (Beijing) Biotechnology Co., Ltd.

[0064] Reagents: Bisphenol A (BPA) (purity >98%), 3-oxoLCA (purity >95%), corn oil (analytical grade);

[0065] Instruments: Fully automated biochemical analyzer, real-time PCR instrument, 16S rRNA sequencing platform.

[0066] Bisphenol A (BPA), also known as 2,2-bis(p-hydroxyphenyl)propane, is a widely used industrial compound with the molecular formula C6H2O. 15 H 16O2. BPA is widely used in the manufacturing processes of canned food and beverage packaging, baby bottles, water bottles, sealants for dental fillings, eyeglass lenses, and hundreds of other everyday products. Although BPA is classified as a low-toxicity substance, its potential harm to pregnant mothers and fetuses cannot be ignored. Studies have shown that BPA exposure can mimic female hormones, disrupting the endocrine system and potentially causing fertility problems, precocious puberty, affecting breast development and reproductive function, and even leading to hormone-related cancers. Furthermore, BPA can induce maternal oxidative stress and cause fetal developmental abnormalities by affecting the expression of UGT family genes (such as UGT1A1, UGT1A6, UGT1A9, and UGT2B1) in the placenta and fetal rat liver.

[0067] In a specific embodiment of this application, BPA treatment of pregnant mice is used to induce maternal oxidative stress in order to construct a pregnancy oxidative stress model.

[0068] 2. Experimental treatment and sample collection

[0069] (1) Pairing and pregnancy confirmation

[0070] Male and female mice were housed together overnight at a 1:2 ratio. The next day, the male and female mice were separated, and pregnancy was confirmed by examining the vaginal plug or sperm (GD1).

[0071] (2) Experimental grouping and treatment plan:

[0072] Control (CON) group (n=10): corn oil (0.2 mL / animal / day);

[0073] Model (BPA) group (n=10): corn oil (0.2 mL / animal / day) + BPA (200 mg / kg BW / day);

[0074] Intervention (BPA+3-oxoLCA) group (n=10): corn oil (0.2 mL / animal / day) + BPA (200 mg / kg BW / day) + 3-oxoLCA (30 mg / kg BW / day).

[0075] All confirmed pregnant female mice were fed a basal reproductive diet. Corn oil, BPA, and / or 3-oxoLCA were administered orally once daily for 18.5 days of gestation; samples were collected by euthanizing the mice via cervical dislocation on day 18.5 of confirmed pregnancy. See the detailed experimental design for more information. Figure 1 .

[0076] Example 2: Study on the effects of 3-oxoLCA on pregnant rats under oxidative stress

[0077] 1. Sample Collection and Analysis

[0078] (1) Sample collection from female mice

[0079] Blood from the mother mouse (blood was collected from the carotid artery on day 18.5 of gestation, and serum was separated), placental tissue, and feces.

[0080] (2) Detection indicators

[0081] Serum oxidative stress indicators: The levels of superoxide dismutase (SOD) (catalog number: A001-3-2) and malondialdehyde (MDA) (catalog number: A003-1-2) in serum samples were detected using commercially available test kits according to the instructions. The kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd., and the determination methods for the indicators were in accordance with the instructions for use of the kits.

[0082] Serum inflammatory factors: Serum levels of interleukin-6 (IL-6) (catalog number: YJ098430), interleukin-8 (IL-8) (catalog number: YJ063162), and interleukin-10 (IL-10) (catalog number: YJ037873) were detected using an enzyme-linked immunosorbent assay (ELISA) kit. The kits were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., and the assay methods were performed according to the kit's instructions.

[0083] Gene expression: Total RNA was extracted from placental and liver samples from maternal mice using TRIzol reagent (Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer's instructions. RNA concentration and A260 / A280 ratios were determined using an Epoch microplate spectrophotometer (BioTek Instruments, Inc., VT, USA), with ratios between 2.0 and 2.2 reserved for further analysis. Reverse transcription was performed using the M5 Sprint qPCR RT kit and gDNA removal agent (Mei5 Biotechnology, Co., Ltd., Beijing, China) according to the manufacturer's protocol. Quantitative RT-PCR was performed using the QuantStudio™ RT-PCR system (Biorad) and SYBR Green reagent (Biorad, CA, USA). Primer sequences are shown in Table 1.

[0084] Table 1. RT-qPCR Primer Sequence List

[0085]

[0086] Gut microbiota: 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, free of contaminants), intact bands (undegraded) on 1% agarose gel, and a concentration ≥50 ng / μL was used as a template for PCR amplification of the V3–V4 hypervariable region of the bacterial 16S rRNA gene using specific primers 338F (5'-ACTCCTACGGGAGGCAGAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were purified (oxygen Biosciences, Union City, CA, USA), quantified, and used for library construction. Paired-end sequencing (2×250bp) was performed on the Illumina MiSeq platform. Noise reduction of the raw sequencing data was performed using DADA2 (version 1.26.0) in QIIME2 (v2022.2), and amplicon sequence variants (ASVs) were generated using default parameters. These were then classified and annotated using the SILVA 138.2 reference database. For downstream analysis, sequences were first refined to the minimum sequence number per sample. Subsequently, analyses such as classification, community diversity (α / β-diversity), species differences (e.g., ANOSIM), and functional prediction (e.g., PICRUSt2) were performed using the Majorbio cloud platform (https: / / cloud.majorbio.com / ). Furthermore, principal coordinate analysis (PCoA) based on bray-curtis, weighted UniFrac, and unweighted UniFrac distances was used to assess β-diversity.

[0087] Bile acid-related transport pathways: Based on 16S rRNA gene data, PICRUSt2 was used for functional prediction, and KEGG pathway annotation was used to infer functional profiles, namely, the functional abundance prediction of 7α-HSDH and BASS family.

[0088] 2. Experimental Results

[0089] (1) Pregnancy status of female mice

[0090] Upon examination, sperm were found in the vaginas of all 30 female mice, meaning that all 30 female mice were pregnant.

[0091] (2) Statistical results of body weight and average daily food intake of female rats during the experimental period

[0092] Figure 14 This study demonstrates the changes in the weight of the mother mice throughout the entire experimental period. Figure 14 A) and average daily feed intake ( Figure 14 B) System monitoring results. Statistical analysis showed that the BPA-exposed group and the control group had similar weight ( P >0.05) and daily food intake ( P No significant differences were observed in terms of >0.05. Furthermore, the intervention group supplemented with 3-oxoLCA did not show significant differences in body dynamics and eating behavior indicators compared to the baseline or BPA exposure groups. P >0.05), indicating that 3-oxoLCA had no significant disturbance effect on metabolic homeostasis and feeding regulation pathways in female mice within the experimental dose range.

[0093] (3) Confirmation of oxidative stress and inflammation in female rats

[0094] Figure 2 The study revealed the detection results of core antioxidant and key inflammatory markers in the serum of pregnant maternal mice. Compared with the control group (CON), the serum of maternal mice in the bisphenol A (BPA) exposure group showed significant characteristics of oxidative stress and inflammatory imbalance. The activity of superoxide dismutase (SOD), the body's primary antioxidant enzyme, was significantly downregulated, indicating that BPA exposure weakened the ability of maternal mice to scavenge superoxide anion free radicals, directly disrupting the oxidation-antioxidant balance. Simultaneously, the concentration of malondialdehyde (MDA), a marker of lipid peroxidation damage, was significantly upregulated, suggesting that free radical accumulation had caused oxidative damage to biomembranes, further supporting the generation of oxidative stress. The significant upregulation of the pro-inflammatory cytokine interleukin-6 (IL-6) reflects the effects of BP. BPA exposure induces oxidative stress and activates inflammatory responses. When 3-oxoLCA was supplemented, the abnormal changes induced by BPA were effectively reversed. Specifically, serum SOD activity was significantly higher than that in the BPA group (meaning that the antioxidant capacity of the pregnant mice was restored and could efficiently clear excess free radicals again), MDA concentration was reduced (indicating that the degree of oxidative damage to biofilms was reduced), and IL-6 level was significantly decreased (suggesting that the inflammatory response was effectively suppressed). In summary, the results clearly suggest that 3-oxoLCA supplementation can improve the serum oxidative stress disorder and inflammatory imbalance in pregnant mice caused by BPA exposure in multiple dimensions by restoring antioxidant enzyme activity, reducing oxidative damage, and inhibiting the release of inflammatory factors.

[0095] (4) Expression of genes related to antioxidant, inflammation and function in maternal rat placenta

[0096] Figure 3The study revealed the mRNA expression of key genes in the placental antioxidant pathway in pregnant mice. Compared to the control group (CON), the bisphenol A (BPA) exposure group (BPA) showed significantly abnormal gene expression: the mRNA expression of Keap1 (a negative regulator of the antioxidant pathway) and NQO1 (an antioxidant compensation gene) was significantly upregulated, while the mRNA expression of Nrf2 (a core transcription factor in the antioxidant pathway) and SOD1 (a key intracellular antioxidant enzyme gene) was significantly downregulated. This change indicates that BPA exposure weakens the activation capacity of the placental antioxidant pathway by enhancing the inhibitory effect of Keap1 on Nrf2, leading to a reduction in the synthesis of core antioxidant enzymes. The upregulation of NQO1 is a passive compensatory response of the placenta to oxidative damage, but it cannot reverse the overall decline in antioxidant capacity, directly reflecting that the placenta is under oxidative stress. Supplementing with 3-oxoLCA can reverse some of the adverse effects of BPA, specifically by significantly downregulating Keap1 mRNA expression and significantly upregulating SOD1 mRNA expression, suggesting that it can restore placental antioxidant defense function by relieving the inhibition of the Nrf2 pathway and promoting the synthesis of antioxidant enzymes.

[0097] Figure 4 The results of placental inflammatory gene mRNA expression analysis showed that the expression of the pro-inflammatory cytokine IL-6 was significantly upregulated in the bisphenol A (BPA) exposure group. Combined with... Figure 3 The oxidative stress caused by the downregulation of SOD1 indicates that placental oxidative damage induced by BPA exposure further activates the placental inflammatory response; while the 3-oxoLCA supplementation group showed a downregulation trend of IL-6, indicating that 3-oxoLCA has a potential inhibitory effect on the local inflammatory response of the placenta while restoring antioxidant capacity.

[0098] Figure 5 The results of placental functional gene mRNA expression analysis showed that, compared with the control group, the bisphenol A (BPA) exposure group significantly downregulated the expression of IGF2 (a key gene regulating placental nutrient transport and angiogenesis), suggesting that BPA exposure impairs the placenta's nutrient supply capacity. Supplementation with 3-oxoLCA not only reversed the inhibitory effect of BPA on IGF2 but also significantly upregulated the expression of syncytinA (a core gene mediating the formation of the placental syncytiotrophoblast and maintaining maternal-fetal barrier function). This indicates that 3-oxoLCA can improve the expression of functional genes, thereby restoring placental nutrient transport efficiency and maintaining its structural integrity, thus ensuring maternal-fetal material exchange and pregnancy stability.

[0099] (5) Expression of genes related to antioxidant, inflammation and bile acid metabolism in the liver of female rats

[0100] Figure 6The results showed the mRNA expression of key genes in the hepatic antioxidant pathway in pregnant mice. Compared with the control group (CON), the bisphenol A (BPA) exposure group (BPA) exhibited significantly abnormal gene expression: the mRNA expression of Keap1 (a negative regulator of the antioxidant pathway) and NQO1 (an antioxidant compensation gene) was significantly upregulated, while the mRNA expression of Nrf2 (a core transcription factor in the antioxidant pathway), CAT (catalase gene, which scavenges hydrogen peroxide), and SOD1 (superoxide dismutase gene, which scavenges superoxide anion) was significantly downregulated. This indicates that BPA exposure, by enhancing the inhibitory effect of Keap1 on Nrf2, blocked the activation of the hepatic antioxidant pathway, leading to a reduction in the synthesis of core antioxidant enzymes; while the upregulation of NQO1 is a passive compensation of the liver in response to oxidative damage, it cannot compensate for the overall decline in antioxidant capacity, directly reflecting that the liver is under oxidative stress. Supplementing with 3-oxoLCA can reverse some of the adverse effects of BPA. It not only significantly downregulated Keap1 mRNA expression and significantly upregulated SOD1 mRNA expression, but also showed an upregulation trend of Nrf2 mRNA expression, suggesting that it can restore the liver's antioxidant defense function by relieving Nrf2 pathway inhibition and promoting the synthesis of antioxidant enzymes.

[0101] Figure 7 The results of liver inflammation gene mRNA expression analysis showed 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) was significantly upregulated in the bisphenol A exposure group. Figure 6 The occurrence of oxidative stress in the liver indicates that BPA-induced oxidative damage further activates inflammation; while 3-oxoLCA supplementation can significantly downregulate IL-6 mRNA expression, indicating that it can effectively inhibit local inflammatory response and reduce inflammatory damage while alleviating liver oxidative stress.

[0102] Figure 8 The results of hepatic bile acid metabolism-related gene mRNA expression analysis showed that, compared with the control group (CON) and the bisphenol A exposure group (BPA), 3-oxoLCA supplementation significantly upregulated the mRNA expression of FXR (bile acid nuclear receptor, regulating bile acid synthesis and transport) and CYP27A1 (the rate-limiting enzyme in bile acid synthesis, catalyzing the conversion of cholesterol to bile acids). This suggests that 3-oxoLCA can regulate the balance of hepatic bile acid synthesis and metabolism by activating the FXR signaling pathway and promoting CYP27A1 expression, and this effect may be one of the important mechanisms by which it improves hepatic oxidative stress and inflammation.

[0103] (6) Intestinal flora of female rat feces

[0104] The microbial community structure in intestinal contents was analyzed using 16S rRNA sequencing. Specific analytical indicators and results are described below:

[0105] At the level of microbial diversity Figure 9 Analysis of α-diversity (Simpson index) in the 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.

[0106] 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.

[0107] (7) Bile acid metabolism pathway of fecal microorganisms in female rats

[0108] 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.

[0109] The main difference between the two groups was in the bile acid transport pathway: the bisphenol A exposure group showed a significant downregulation of bile acids: Na+ + Cotransporter (BASS family) pathway function Figure 11 B). The BASS family is the core carrier for the absorption and transport of bile acids in the intestine. Downregulation of its pathway function directly inhibits the enterohepatic circulation of bile acids, which can lead to impaired bile acid absorption, increased burden on liver synthesis, and even indirectly exacerbate liver oxidative-inflammatory damage. However, the 3-oxoLCA supplementation group did not show a significant effect on this transport pathway, suggesting that 3-oxoLCA can maintain normal bile acid transport function by avoiding the inhibition of the BASS family pathway by bisphenol A exposure, thus providing a basis for repairing bile acid metabolism disorders and protecting liver function.

[0110] Example 3: Study on the effects of 3-oxoLCA on offspring of oxidative stress-exposed female rats.

[0111] (1) Fetal weight

[0112] At 18.5 days of gestation, the mother mice were euthanized according to animal ethics guidelines. After confirming the absence of vital signs, the dissection was performed. The mother mouse was fixed to the dissection table with her abdomen facing upwards. The abdominal skin was disinfected with 75% alcohol. The skin and abdominal muscles were cut along the midline of the abdomen to expose the uterine horn. The uterus and surrounding connective tissue were carefully separated and removed intact, and placed in sterile saline. Next, the uterine wall was gently torn open with forceps, and each fetus was removed. After removing the fetal membranes and umbilical cord, the fetuses were rinsed in fresh sterile saline to remove residual blood and tissue debris. Finally, the fetuses were weighed: the electronic balance was adjusted to 0.001g and calibrated. Each fetus was individually dried with sterile filter paper and quickly placed in the center of the weighing pan. After the reading stabilized, the weight was recorded. This process was repeated to weigh all fetuses, and the number of fetuses was recorded simultaneously. The average weight of fetuses in the litter was calculated for subsequent intergroup difference analysis.

[0113] As shown in Table 2, compared with the control group (CON), the fetal litter weight (reflecting the overall growth level of fetuses in the litter) and average fetal weight (reflecting the average growth status of individual fetuses) were significantly lower in the bisphenol A (BPA) exposure group. This indicates that BPA exposure has a significant inhibitory effect on intrauterine fetal growth, leading to reduced fetal growth performance. Supplementation with 3-oxoLCA effectively improved this situation, specifically by significantly increasing the average fetal weight. This suggests that 3-oxoLCA supplementation can specifically alleviate the inhibitory effect of BPA exposure on individual fetal growth and, to some extent, reverse the decrease in average fetal weight caused by BPA, providing experimental evidence for improving the intrauterine growth environment for fetuses.

[0114] Table 2 Effects of 3-oxoLCA on BPA-induced reproductive performance in pregnant female mice

[0115]

[0116] Note: Different superscripts for a, b, and c indicate significant differences. P <0.05). IUGR: Intrauterine growth restriction.

[0117] (2) Expression of antioxidant and inflammation-related genes in the liver of young rats

[0118] Liver samples were collected from offspring mice, and the mRNA expression of antioxidant and inflammation-related genes was analyzed using the detection methods described in Example 1. The results showed that bisphenol A (BPA) exposure had a significant cross-generational effect on gene expression in the livers of offspring mice. Compared with the control group (CON), the mRNA expression of the antioxidant gene SOD2 (mitochondrial superoxide dismutase, responsible for scavenging mitochondrial-derived free radicals) was significantly downregulated in the BPA-exposed group, indicating that BPA exposure weakens the antioxidant defense capacity at the mitochondrial level in the livers of offspring mice and increases the risk of hepatic oxidative stress. Supplementation with 3-oxoLCA effectively reversed this abnormality, significantly upregulating SOD2 mRNA expression in the livers of offspring mice, suggesting that it can enhance the ability of the livers of offspring mice to cope with oxidative damage by restoring the expression of mitochondrial antioxidant enzyme genes (see Example 1). Figure 12 ).

[0119] Figure 13 The results showed that BPA exposure significantly increased the gene expression level of the pro-inflammatory factor IL-8 in the liver of offspring mice. P <0.05), while after supplementing the diet of female mice with 3-oxoLCA, this index significantly decreased to levels close to those of the control group ( P <0.05). IL-8 is a core signaling molecule in the inflammatory response, and its elevated expression usually indicates tissue damage and immune abnormalities. The above results suggest that 3-oxoLCA can effectively inhibit BPA-induced liver inflammation and has potential anti-inflammatory protective effects.

Claims

1. The use of 3-oxoLCA in the preparation of a medicament / composition for alleviating decreased fetal growth performance caused by maternal oxidative stress during pregnancy, characterized in that, Compared to fetuses born to mothers who did not experience oxidative stress, the reduced fetal growth performance was manifested in decreased litter weight and average weight.

2. The use according to claim 1, characterized in that, The gestating mother is a pregnant animal.

Images