Use of baicalein in the preparation of a drug for treating and / or alleviating preeclampsia

Abstract

This invention relates to the use of baicalin in the preparation of drugs for treating and / or alleviating preeclampsia. The drugs are used to inhibit ferroptosis in placental trophoblasts and / or placental tissue, manifested by increasing the expression level of at least one of GPX4, SLC7A11, and FTH1, increasing GSH content and / or GPX activity, and decreasing Fe... 2+ It can reduce MDA, lipid ROS and / or mitochondrial ROS levels, and improve preeclampsia-related hypertension, proteinuria, placental oxidative stress and adverse pregnancy outcomes. This application can be used to prepare preeclampsia treatment and / or alleviation drugs with baicalin monomer as the active ingredient.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology, specifically relating to the application of baicalin in the preparation of drugs for treating and / or alleviating preeclampsia. Background Technology

[0002] Preeclampsia is a pregnancy-specific disorder that occurs after 20 weeks of gestation, characterized by new-onset hypertension with or without proteinuria and multiple organ dysfunction. It can lead to placental abruption, fetal growth restriction, premature birth, maternal organ damage, and even maternal and infant death. Current clinical management mainly focuses on lowering blood pressure, relieving spasms, monitoring, and termination of pregnancy if necessary. There is still a lack of effective drugs targeting the pathological mechanisms of the placenta.

[0003] A growing body of research indicates that placental ischemia and hypoxia, increased oxidative stress, abnormal lipid peroxidation, and iron metabolism disorders are closely related to the development and progression of preeclampsia. Ferropreservation is an iron-dependent form of cell death characterized by excessive accumulation of lipid peroxides, imbalance of the antioxidant system, and elevated intracellular free iron. In preeclampsia, abnormal activation of ferroptosis in trophoblasts and placental tissue is considered a significant factor contributing to placental dysfunction.

[0004] Baicalein is a flavonoid monomeric compound derived from Scutellaria baicalensis, possessing biological functions such as antioxidant, anti-inflammatory, and regulation of iron homeostasis. While existing technologies have reported protective effects of baicalein against nervous system damage, oxidative stress-related diseases, and some pregnancy-related diseases, and studies have also reported on the use of other natural small molecules in the intervention of preeclampsia or ferroptosis, there is a lack of publicly available and systematically experimentally verified uses of baicalein in the treatment and / or relief of preeclampsia, particularly through its role in inhibiting ferroptosis in placental trophoblast cells and / or placental tissue.

[0005] Therefore, there is still a need for a preeclampsia drug use plan that is supported by experimental data and targets the abnormal activation of placental oxidative stress and ferroptosis. Summary of the Invention

[0006] Technical problems to be solved The present invention aims to provide the application of baicalin in the preparation of drugs for treating and / or alleviating preeclampsia, in order to solve the problem of the lack of effective intervention methods in the prior art for the key pathological link of abnormal activation of placental ferroptosis.

[0007] Technical solution To address the aforementioned technical problems, this invention provides the use of baicalin monomer as an active ingredient in the preparation of drugs for treating and / or alleviating preeclampsia.

[0008] Preferably, the drug is used to inhibit ferroptosis of placental trophoblasts and / or placental tissue.

[0009] Preferably, the inhibition of ferroptosis is manifested by increasing the expression level of at least one of GPX4, SLC7A11 and FTH1.

[0010] Preferably, the inhibition of ferroptosis manifests as a reduction in free Fe. 2+ The level of at least one of MDA, lipid ROS and mitochondrial ROS, and / or increase at least one of GSH content and GPX activity.

[0011] Preferably, the treatment and / or relief are manifested as a reduction in blood pressure and / or urinary protein levels.

[0012] Preferably, the treatment and / or relief are manifested as improvement in placental oxidative stress and / or pregnancy outcomes, wherein the improvement in pregnancy outcomes includes increased live births, improved fetal developmental indicators (including fetal mouse / placental weight ratio) and / or reduced embryo resorption rate.

[0013] Preferably, baicalein is the sole active ingredient in the drug; the drug also includes pharmaceutically acceptable excipients.

[0014] Preferably, the drug is an oral preparation.

[0015] Beneficial effects Compared with the prior art, the present invention has at least the following beneficial effects: (1) Examples show that baicalin has an inhibitory effect on placental trophoblast and placental tissue ferroptosis, and forms an experimental data chain of "compound-mechanism indicator-maternal phenotype-pregnancy outcome".

[0016] (2) Baicalein can reduce lipid ROS, mitochondrial ROS, and free Fe in an in vitro hypoxic trophoblast cell model. 2+ It increased GSH content, GPX activity, and expression of anti-ferroptosis-related proteins such as GPX4 and SLC7A11 to varying degrees, along with MDA levels.

[0017] (3) Baicalein can reduce blood pressure and urinary protein levels in L-NAME-induced preeclampsia animal models, improve placental oxidative stress and pregnancy outcomes, and simultaneously improve placental tissue ferroptosis-related indicators.

[0018] (4) The application scheme of the present invention uses abnormal activation of placental ferroptosis as an intervention step, providing experimental basis for the preparation of preeclampsia drugs with baicalin monomer as active ingredient. Attached Figure Description

[0019] Figure 1 Venn diagram of the intersection targets of baicalin, preeclampsia, and ferroptosis.

[0020] Figure 2PPI network diagram and core target analysis diagram of the intersection targets of baicalin, preeclampsia and ferroptosis.

[0021] Figure 3 The images show the GO function enrichment results and KEGG pathway enrichment results for the intersection targets.

[0022] Figure 4 The diagram shows the molecular docking results of baicalin with the core target and the molecular dynamics simulation results of the protein-ligand complex.

[0023] Figure 5 The figure shows the results of baicalin alleviating hypoxia-induced oxidative stress and mitochondrial damage in HTR-8 / SVneo cells. Figure 5 A shows the C11-BODIPY method for detecting lipid ROS. Figure 5 B shows the MitoSOX method for detecting mitochondrial ROS. Figure 5 C shows the mitochondrial morphology observed under a transmission electron microscope. Figure 5 D shows the flow cytometry detection of mitochondrial membrane potential.

[0024] Figure 6 The image shows the results of baicalin alleviating hypoxia-induced ferroptosis in HTR-8 / SVneo cells. Figure 6 A to Figure 6 D represents free Fe. 2+ MDA, GSH and GPX activity, Figure 6 E shows the results of the Western blot analysis. Figure 6 F to Figure 6 H represents the expression of SLC7A11, GPX4, and FTH1 proteins, respectively.

[0025] Figure 7 The figure shows the results of baicalin improving fetal development in L-NAME-induced preeclampsia mice. Figure 7 A shows the external appearance of the uterus, fetus, and placenta. Figure 7 B indicates embryo absorption rate. Figure 7 C indicates the number of live births. Figure 7 D indicates the ratio of fetal mouse weight to placental weight.

[0026] Figure 8 This is a diagram showing the results of baicalin inhibiting L-NAME-induced placental ferroptosis in preeclampsia mice. Figure 8 A shows the expression of SLC7A11, GPX4, and FTH1 proteins. Figure 8 B indicates placental GSH levels. Figure 8 C indicates the placental MDA level. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited to the following embodiments. Equivalent substitutions or conventional changes made by those skilled in the art based on the present invention without departing from its essence should fall within the scope of protection of the present invention.

[0028] The baicalein referred to in this invention is the compound 5,6,7-trihydroxyflavone, and the technical solution of this invention uses baicalein monomer as the active ingredient. The baicalein used in the embodiments of this invention was purchased from MedChemExpress (MCE), product code HY-N0196. Unless otherwise stated, the raw materials, reagents, and instruments used in the following embodiments can be obtained through conventional commercial channels; unless otherwise specified, the experimental methods in the embodiments are conventional methods in the art.

[0029] Unless otherwise stated, GPX4 is glutathione peroxidase 4, SLC7A11 is solute carrier family 7 member 11, FTH1 is ferritin heavy chain 1, GSH is reduced glutathione, GPX is glutathione peroxidase, MDA is malondialdehyde, ROS is reactive oxygen species, and L-NAME is Nω-nitro-L-arginine methyl ester.

[0030] Unless otherwise stated, experimental data are expressed as mean ± standard deviation. Comparisons between two groups were performed using t-tests or Wilcoxon nonparametric tests based on data distribution and homogeneity of variance; comparisons among multiple groups were performed using one-way ANOVA or Kruskal-Wallis tests based on data distribution and homogeneity of variance. p < 0.05 indicated statistical significance.

[0031] Example 1: Analysis of potential targets and pathways of baicalin Potential targets of baicalin were collected using the TCMSP database, the SwissTargetPrediction platform, and the PharmMapper database. After UniProt normalization and deduplication, 589 baicalin-related targets were obtained. 2495 preeclampsia-related targets were collected using the GeneCards and OMIM databases, and 564 ferroptosis-related targets were collected using the FerrDb database. Intersection analysis of these three databases yielded 29 overlapping targets.

[0032] The 29 intersection target points were imported into the STRING database to construct a PPI network, resulting in an interaction network with 29 nodes and 181 edges. The average node degree was 12.483, the clustering coefficient was 0.371, and the network density was 0.223. The core target points mainly include ALB, TP53, HIF1A, PPARG, PTGS2, GSK3B, EGFR, TXN, SRC, SIRT1, and RELA.

[0033] Table 1 Core Target Information Further GO and KEGG enrichment analysis revealed that the overlapping targets mainly involve biological processes such as cellular response to hypoxia, oxidative stress, and cellular redox homeostasis, with related pathways including the HIF-1 signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, glutathione metabolism pathway, and ferroptosis pathway.

[0034] Table 2. Molecular docking results of baicalin with some core targets. Molecular docking results showed that, among the core targets detected, baicalin exhibited low calculated binding energies with AR and PTGS2. Molecular dynamics simulations revealed that the AR-baicalin and PTGS2-baicalin complexes were stable under simulated conditions. These results suggest that baicalin may participate in the regulation of preeclampsia-related ferroptosis through multiple targets and pathways.

[0035] Example 2: Inhibitory effect of baicalin on hypoxia-induced trophoblast ferroptosis HTR-8 / SVneo human feeder cells were cultured in RPMI-1640 complete medium. Normoa conditions were 95% air, 5% CO2, and 37°C; hypoxia conditions were 94% N2, 1% O2, 5% CO2, and 37°C.

[0036] Cells were divided into four groups: control group, hypoxia group, hypoxia + 5 μM baicalein group, and hypoxia + 20 μM baicalein group. Lipid ROS, mitochondrial ROS, and free Fe were measured. 2+ The study included GSH content, GPX activity, MDA content, and the expression of GPX4, SLC7A11, and FTH1 proteins.

[0037] Wherein, lipid ROS refers to the lipid reactive oxygen species level measured by the C11-BODIPY method, mitochondrial ROS refers to the mitochondrial reactive oxygen species level measured by the MitoSOX method, and free Fe 2+ The intracellular free ferrous ion level was determined using calcein-AM fluorescence spectrometry; GSH was reduced glutathione, GPX was glutathione peroxidase, and MDA was malondialdehyde. GSH, GPX activities, and MDA were detected using the corresponding biochemical reagent kits; the expression of GPX4, SLC7A11, and FTH1 proteins was detected by Western blot.

[0038] The results showed that hypoxia treatment induced oxidative stress and ferroptosis-related changes in HTR-8 / SVneo cells, manifested as increased lipid ROS and mitochondrial ROS, increased MDA, decreased GSH content and GPX activity, and downregulated expression of GPX4, SLC7A11, and FTH1. Baicalein intervention had varying degrees of regulatory effects on these indicators. Specifically, 5 μM and 20 μM baicalein both reduced lipid ROS, mitochondrial ROS, and MDA levels, increased GPX activity, and partially increased GPX4 and SLC7A11 expression; 5 μM baicalein reduced free Fe... 2+ At the same level, 20 μM baicalein had a more significant effect on increasing GSH levels.

[0039] Table 3 Grouping and treatment conditions of in vitro models Table 4. Results of in vitro oxidative stress-related indicators (mean ± standard deviation) Table 5. Results of in vitro ferroptosis-related indicators (mean ± standard deviation) The above results indicate that baicalin can inhibit hypoxia-induced trophoblast ferroptosis, thus providing in vitro experimental evidence for its use in the treatment and / or relief of preeclampsia.

[0040] Example 3: The ameliorative effect of baicalin on L-NAME-induced preeclampsia in mice Eight-week-old ICR female mice were mated with male mice at a 2:1 ratio. The appearance of vaginal plugs was recorded as day 0.5 of gestation. From day 8.5 of gestation, a preeclampsia model was established using L-NAME. Pregnant mice were randomly divided into a blank control group, an L-NAME group, an L-NAME + aspirin group, an L-NAME + 50 mg / kg / d baicalein group, and an L-NAME + 100 mg / kg / d baicalein group.

[0041] The blank control group received a subcutaneous injection of normal saline in the neck and back on day 8.5 of pregnancy, and 0.5% CMC-Na was administered by gavage starting on day 12.5 of pregnancy; the L-NAME group received a subcutaneous injection of 125 mg / kg / d L-NAME in the neck and back on day 8.5 of pregnancy, and 0.5% CMC-Na was administered by gavage starting on day 12.5 of pregnancy; the aspirin group received 15 mg / kg / d aspirin by gavage starting on day 12.5 of pregnancy; and the baicalein group received 50 mg / kg / d or 100 mg / kg / d baicalein by gavage starting on day 12.5 of pregnancy.

[0042] Blood pressure was measured on days 8.5, 12.5, and 17.5 of gestation. Urine was collected to detect urinary protein. Mice were sacrificed on day 17.5 of gestation, and the number of live offspring, fetal development indicators (including fetal mouse / placental weight ratio), embryo resorption rate, and other indicators were recorded. The expression of GSH, MDA, GPX4, SLC7A11, and FTH1 in placental tissue was also detected.

[0043] The results showed that L-NAME could induce a preeclampsia-like phenotype, characterized by elevated blood pressure, elevated proteinuria, restricted weight gain, reduced live birth count, decreased fetal developmental indicators (including fetal mouse / placental weight ratio), and increased embryo resorption rate. After intervention with baicalein, these abnormalities were improved to varying degrees, and GSH levels increased, MDA levels decreased, and GPX4, SLC7A11, and FTH1 expression was upregulated in placental tissue.

[0044] Table 6. In vivo model grouping and dosing regimen Table 7. Maternal phenotypic test results (mean ± standard deviation) Table 8. Fetal outcome test results (mean ± standard deviation) Table 9. Results of placental tissue ferroptosis-related indicators (mean ± standard deviation) The above results indicate that baicalin can improve maternal phenotype, fetal outcome, and placental ferroptosis-related indicators in preeclampsia animal models, thus supporting its use in the preparation of drugs for the treatment and / or relief of preeclampsia.

[0045] Example 4: Summary of Results As demonstrated in Examples 1 to 3, baicalin has an interventional effect on oxidative stress and abnormal activation of ferroptosis associated with preeclampsia. This effect is reflected in changes at the cellular level in lipid ROS, mitochondrial ROS, Fe2+, GSH, GPX activity, MDA, and related protein expression, as well as in improvements at the animal level in blood pressure, urinary protein, placental ferroptosis-related indicators, and fetal outcomes.

[0046] Therefore, baicalin monomers can be used as active ingredients in the preparation of drugs for the treatment and / or relief of preeclampsia, especially suitable for preeclampsia accompanied by placental oxidative stress and / or abnormal activation of placental ferroptosis.

Claims

1. The use of baicalin in the preparation of drugs for treating and / or alleviating preeclampsia, characterized in that, The baicalein is 5,6,7-trihydroxyflavone, and the drug is used to inhibit ferroptosis in placental trophoblasts and / or placental tissue.

2. The application according to claim 1, characterized in that, The inhibition of ferroptosis is manifested by increasing the expression level of at least one of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and ferritin heavy chain 1 (FTH1).

3. The application according to claim 1 or 2, characterized in that, The inhibition of ferroptosis is manifested by reducing the level of at least one of free ferrous ions (Fe2+), malondialdehyde (MDA), lipid reactive oxygen species, and mitochondrial reactive oxygen species.

4. The application according to any one of claims 1 to 3, characterized in that, The inhibition of ferroptosis is manifested by increasing the content of reduced glutathione (GSH) and / or the activity of glutathione peroxidase (GPX).

5. The application according to any one of claims 1 to 4, characterized in that, The treatment and / or relief are manifested in lowering blood pressure and / or urinary protein levels.

6. The application according to any one of claims 1 to 5, characterized in that, The treatment and / or relief are manifested in improved placental oxidative stress and / or pregnancy outcomes.

7. The application according to claim 6, characterized in that, The improvements in pregnancy outcomes include increasing the number of live births, improving fetal development indicators (including fetal mouse / placental weight ratio), and / or reducing embryo resorption rate.

8. The application according to any one of claims 1 to 7, characterized in that, Baicalein is the only active ingredient in the drug.

9. The application according to claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients, and the drug is an oral formulation.

10. The application according to any one of claims 1 to 9, characterized in that, The term "preeclampsia" refers to preeclampsia accompanied by placental oxidative stress and / or abnormal activation of placental ferroptosis.

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