Application of 6-gingerol in the preparation of a drug for treating preeclampsia

Abstract

The present invention relates to the use of 6-gingerol in the preparation of a medicament for treating preeclampsia, belonging to the technical field of medicine. The present invention provides the use of 6-gingerol in the preparation of a medicament, health food or special medical food for treating preeclampsia. 6-Gingerol blocks the initiation of mitophagy by reducing the expression of BNIP3 and LC3, or regulates H / R-induced mitophagy by inhibiting the production of mitochondrial ROS, thereby playing a role in treating or preventing preeclampsia medicaments. The present invention discovers for the first time that 6-gingerol can block the initiation of mitophagy by reducing the expression of BNIP3 and LC3 or regulate H / R-induced mitophagy by inhibiting the production of mitochondrial ROS, so as to achieve the purpose of treating or preventing preeclampsia.

Description

Technical Field

[0001] The present invention relates to the use of 6-gingerol in the preparation of a medicament for treating preeclampsia, and belongs to the field of pharmaceutical technology. Background Art

[0002] Preeclampsia (PE) is a common pregnancy complication and a major cause of morbidity and mortality in pregnant women and fetuses. Approximately 8% of pregnancies worldwide are affected by PE. The placenta is the direct connection between the mother and the fetus and plays the most important role in the exchange of oxygen and nutrients. Therefore, normal placental development is essential for fetal growth and development. During embryo implantation, extravillous trophoblasts invade the maternal decidua and spiral arteries, leading to the remodeling of spiral arteries. Any obstruction in this process resulting in insufficient placental function can lead to pregnancy complications such as PE.

[0003] Mitochondria are involved in redox homeostasis, Ca 2+ homeostasis, apoptosis, and various other cellular processes and are very sensitive to hypoxia and oxidative stress. Studies have shown that improper arterial remodeling leading to placental hypoplasia-induced ischemia and excessive ROS are closely related to mitochondrial dysfunction during the PE process. Mitophagy is a form of autophagy within cells and is defined as the selective degradation of damaged or dysfunctional mitochondria by autophagy. Any defect in mitophagy may lead to the production and accumulation of cellular ROS, ultimately resulting in the occurrence of preeclampsia.

[0004] BNIP3 (BCL2 and adenovirus E1B 19-kd interacting protein 3) is involved in receptor-dependent mitotic pathways and acts as an inducer in response to hypoxia and other stimuli. BNIP3 can directly bind to LC3 through its LC3-interacting region, inducing cell death and autophagy. Similar to autophagy, LC3-II, which is converted from LC3-I mediated by P62 and located on the autophagosome membrane, is the main signal during the autophagy process and regulates the digestion of damaged mitochondria by autophagosomes. Previous studies have shown that during the development of PE, the dysregulation of BNIP3 is related to impaired placental autophagy and oxidative stress.

[0005] 6-Gingerol is the main compound extracted from ginger, which has anti-inflammatory, antioxidant, anti-cancer and anti-apoptotic effects. In various ischemia-reperfusion diseases, 6-gingerol can significantly reduce the pro-inflammatory factors in tissues such as intestine, brain and myocardium, and inhibit apoptosis and oxidative stress. In addition, 6-gingerol can effectively improve atherosclerosis and lung injury. For example, Chinese patent document CN111110664A (application number 202010061480.8) discloses the application of 6-gingerol in the preparation of drugs for treating neonatal hypoxic-ischemic encephalopathy. This invention first proves that 6-gingerol can reduce the brain injury after HIE in neonatal mice, inhibit the death of nerve cells after HIE in neonatal mice, significantly reduce the level of Bax protein in the lesioned side cortex after HIE, increase the expression of Bcl-2 protein, and increase the ratio of Bcl-2 / Bax. At the same time, 6-gingerol can reduce the expression of pro-inflammatory factors TNF-α and IL-1β, thereby reducing the inflammatory response after HIE and improving the short-term nerve reflex and long-term motor ability of mice after HIE. Therefore, it can be considered as an effective candidate drug for the neuroprotective treatment of HIE brain injury and has important clinical application value. However, there is no relevant research report on the protective effect of 6-gingerol on placental dysfunction and its related obstetric complications such as preeclampsia. Summary of the Invention

[0006] The present invention aims at the deficiencies of the prior art and provides the application of 6-gingerol in the preparation of drugs for treating preeclampsia.

[0007] The technical solution of the present invention is as follows:

[0008] The application of 6-gingerol in the preparation of drugs for treating preeclampsia.

[0009] Preferably according to the present invention, the active ingredient of the drug for treating preeclampsia contains 6-gingerol.

[0010] Preferably according to the present invention, the drug blocks the initiation of mitophagy by reducing the expression of BNIP3 and LC3 through 6-gingerol.

[0011] Preferably according to the present invention, the drug regulates H / R-induced mitophagy by inhibiting the production of mitochondrial ROS through 6-gingerol.

[0012] The application of 6-gingerol in the preparation of health foods or foods for special medical purposes for preventing preeclampsia.

[0013] Preferably according to the present invention, the active ingredient in the health food or food for special medical purpose contains 6-gingerol.

[0014] Preferably according to the present invention, the health food or food for special medical purpose blocks the initiation of mitophagy by reducing the expression of BNIP3 and LC3 through 6-gingerol.

[0015] Preferably according to the present invention, the health food or food for special medical purposes regulates hypoxia / reoxygenation (H / R)-induced mitophagy by inhibiting the production of mitochondrial reactive oxygen species (ROS) by 6-gingerol.

[0016] Beneficial effects

[0017] The present invention discovers for the first time that 6-gingerol can block the initiation of mitophagy by reducing the expression of BNIP3 and LC3, or regulate H / R-induced mitophagy by inhibiting the production of mitochondrial ROS, thereby achieving the purpose of treating or preventing preeclampsia. Description of the drawings

[0018] Figure 1(A) is a representative image photo of HTR8 cells transfected with RFP-mitochondria and GFP-LC3 plasmids and subjected to hypoxia / reoxygenation treatment in Example 1;

[0019] Wherein: scale bar: 100 μm;

[0020] Figure 1(B) is a representative staining photo of Mito-Tracker and Lyso-Tracker in Example 1;

[0021] Wherein: scale bar: 100 μm;

[0022] Figure 1(C) is a representative photo of the results of Western blots of LC3, P62, BNIP3, and TOMM20 in Example 1;

[0023] Figure 1(D) is a statistical bar graph of Western blots of LC3, P62, BNIP3, and TOMM20 in Example 1;

[0024] Figure 2(A) is a statistical bar graph of the weights of placentas and fetal mice in each group in Example 2;

[0025] Figure 2(B) is a representative image photo of immunohistochemical staining for detecting BNIP3 in placentas of each group in Example 2;

[0026] Wherein: scale bar: 50 μm;

[0027] Figure 2(C) is a photo of Western blot and corresponding semi-quantitative results of BNIP3 in placentas of each group in Example 2;

[0028] Figure 2(D) is a statistical bar graph of Western blot and corresponding semi-quantitative results of BNIP3 in placentas of each group in Example 2; Detailed implementation manners

[0029] The technical solutions of the present invention will be further described below in conjunction with examples, but the scope of protection of the present invention is not limited thereto.

[0030] Preparation of Preeclampsia Mouse Model

[0031] 10-week-old C57BL / 6 female and male mice purchased from the Experimental Animal Center of Shandong University were maintained on a 12 h / 12 h light / dark cycle at 18 - 22 °C and had free access to food and water.

[0032] Female mice were mated with male mice at a ratio of 2:1, and the time when vaginal plugs were found was recorded as gestational day 0.5. Pregnant mice were randomly divided into a CT group (n = 6), an L-NAME group (n = 6), and an L-NAME + 6-gingerol group (n = 6). Mice in the L-NAME group and the L-NAME + 6-gingerol group were subcutaneously injected with L-NAME at a dose of 125 mg / kg / d (MedChemExpress, USA) from GD9.5 to GD18.5. The L-NAME + 6-gingerol group was also intraperitoneally injected with 2 mg / kg 6-gingerol (MedChemExpress, USA) from GD8.5 to GD18.5. The CT group was intraperitoneally and subcutaneously injected with normal saline during the same period. At GD18.5, the mice were sacrificed by cervical dislocation, and the placental weight, the number of fetal mice, and the fetal weight were recorded. The placentas were collected and stored at -80 °C or fixed with 4% paraformaldehyde for further analysis.

[0033] Cell Culture and Treatment

[0034] Human trophoblast HTR8 / Svneo (HTR8) was purchased from the American Type Culture Collection and cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium (Invitrogen, California, USA) supplemented with 10 wt% fetal bovine serum (GIBCO, New Zealand) and 1% penicillin-streptomycin (Solarbio, China). The cells were placed under hypoxic conditions (4% N2 / 5% CO2 / 1% O2, v / v) for 12 h, then reoxygenated (5% CO2 / 95% air, v / v) for 12 h, and then pretreated with 6-gingerol and Mito-TEMPO to establish an in vitro hypoxia / reoxygenation (H / R) model.

[0035] According to the instructions of the manufacturer (GENE, China), RFP-mitochondria and GFP-LC3 plasmids were co-transfected into HTR8 cells to measure the autophagic flux of mitochondria. Twenty-four hours after transfection, Cell images of GFP-LC3 and RFP-mitochondria were captured using a

[0036] Immunohistochemistry

[0037] Mouse placental tissues were fixed in 4 wt% paraformaldehyde solution and embedded in paraffin. After dewaxing and hydration with gradient ethanol, antigen retrieval was performed on the sections. Then, the sections were blocked with goat serum (10 wt%) at room temperature for 30 min. Subsequently, the sections were incubated with the primary antibody (1:200) at 4 °C overnight. After washing with PBS, the placental tissues were incubated with the secondary antibody at 37 °C for 1 h. Photographs were taken and recorded using an inverted fluorescence microscope (Olympus, BX53F, Japan).

[0038] Western Blot analysis

[0039] Cells and tissues were lysed in RIPA buffer (Solarbio, Beijing, China) containing protease inhibitor (Beyotime, Beijing, China), and quantification was performed using a BCA kit (Solarbio, Beijing, China). Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. After soaking in 5 wt% skim milk at room temperature for 1 h, the membranes were incubated with the primary antibody diluted at 1:1000 at 4 °C overnight. The membranes rinsed with TBST were hybridized with horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence detection kit (Amersham LifeScience, Buckinghamshire, UK). The target protein was normalized to β-actin and quantitatively expressed. Independent experiments were performed at least three times, and representative images were presented.

[0040] LC3A / B antibody (12741) was purchased from Cell Signaling Technology (USA). TOM20 (ab56783) antibody was purchased from Abcam (USA). β-Actin (6600901) was purchased from Proteintech (China). Horseradish peroxidase-labeled goat anti-mouse immunoglobulin G (GB23301) and horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (GB23303) were purchased from Servicebio (China).

[0041] Statistical analysis

[0042] Data were expressed as the mean ± standard deviation (SD) of at least three different biological replicates. Student's t-test was used to compare two groups of data. Multiple comparisons (≥3) were performed using one-way analysis of variance (ANOVA), followed by Tukey-Kramer multiple comparison test. The quantitative results were visualized using bar charts. P < 0.05 was considered significant, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0043] Example 1: 6-Gingerol inhibits mitochondrial hyperautophagy and protects mitochondrial function from H / R injury

[0044] Existing studies have shown that mitochondrial damage can induce mitophagy, a selective autophagy that degrades mitochondrial fragments through lysosomes. In this example, the effect of 6-gingerol on H / R injury was investigated. It was found that 6-gingerol could inhibit oxidative stress injury generated by mitochondria due to its antioxidant effect. Furthermore, the effect of the mitochondrial ROS scavenger Mito-TEMPO on mitophagy under H / R conditions was detected. The specific steps are as follows:

[0045] Human trophoblast HTR8 / Svneo (HTR8) was purchased from the American Type Culture Collection and cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium (Invitrogen, USA) containing 10 wt% fetal bovine serum (GIBCO, New Zealand) and 1 wt% penicillin-streptomycin (Solarbio, China). The specific steps were performed according to the product instructions. First, 6-gingerol (10 μM) and Mito-TEMPO (5 μM) were added to the corresponding group of cell culture media to pretreat the cells for 4 h. Then, the cells were cultured under hypoxic conditions (4% N2 / 5% CO2 / 1% O2, volume percentage) for 12 h, followed by reoxygenation (5% CO2 / 95% air, volume percentage) for 12 h to establish an in vitro hypoxia / reoxygenation (H / R) model.

[0046] Then, according to the product instructions (GENE, China), RFP-mitochondria and GFP-LC3 plasmids were added to the HTR8 cell culture medium for co-transfection to track the possible co-localization between mitochondria and autophagosomes. H / R treatment significantly increased the co-localization between mitochondria and LC3-labeled autophagosomes, while 6-gingerol decreased the co-localization, as shown in Figure 1(A).

[0047] To study the fusion of mitophagosomes and lysosomes (the final step of mitophagy), Mito-Tracker and Lyso-Tracker probes were used to label mitochondria and lysosomes, respectively. The specific experimental steps are as follows:

[0048] HTR8 cells were processed and cultured for a specific time. Specifically, 6-gingerol (10 μM) and Mito-TEMPO (5 μM) were added to the corresponding group of cell culture media to pretreat the cells for 4 h. Then, the cells were cultured under hypoxic conditions (4% N2 / 5% CO2 / 1% O2, volume percentage) for 12 h, and then reoxygenated (5% CO2 / 95% air, volume percentage) for 12 h. After that, the cell culture medium was removed, and Mito-Tracker Green staining working solution and Lyso-Tracker Red staining working solution (Beyotime, China) were added, and the cells were co-incubated at 37 °C for 30 minutes. The staining working solution was removed, and fresh cell culture medium was added. Subsequently, a micro confocal system (Molecular Devices, USA) was used for observation. As shown in Figure 1(B), under H / R conditions, the co-localization in the overlapping area of Mito-Tracker and Lyso-Tracker increased significantly, which confirmed the fusion of mitochondria and lysosomes. 6-gingerol pretreatment reduced this fusion. At the same time, the effect of Mito-TEMPO was the same as that of 6-gingerol.

[0049] In addition, in the H / R group, P62 (an autophagy protein) did not decrease, which accumulated due to impaired fusion of autophagosomes and lysosomes, indicating that H / R impaired autophagic flux. 6-gingerol and Mito-TEMPO restored the degradation of P62. In addition, the results of HTR8 cells showed that H / R treatment downregulated the mitochondrial marker TOMM20, upregulated the expression of BNIP3 and the LC3II / LC3I ratio. However, 6-gingerol decreased the expression of BNIP3 and the LC3II / LC3I ratio. As a mitochondrial ROS inhibitor, Mito-TEMPO further identified the effect of mitochondrial ROS on mitophagy under H / R. Western blot results showed that Mito-TEMPO, like 6-gingerol, blocked the initiation of mitophagy by reducing the expression of BNIP3 and LC3, as shown in Figure 1(C) and Figure 1(D). The specific experimental steps are as follows:

[0050] HTR8 cells were treated and cultured for a specific time. 6-gingerol (10 μM) and Mito-TEMPO (5 μM) were added to the corresponding group of cell culture media to pretreat the cells for 4 h. Then the cells were cultured under hypoxic conditions (4% N2 / 5% CO2 / 1% O2, v / v) for 12 h, followed by reoxygenation (5% CO2 / 95% air, v / v) for 12 h. After removing the cell culture medium, the cells were lysed in RIPA buffer (Solarbio, Beijing, China) containing protease inhibitor (Beyotime, Beijing, China), and protein quantification was performed using a BCA kit (Solarbio, Beijing, China). The specific steps were carried out according to the product instructions. Equal amounts of protein were separated on an SDS-PAGE gel and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. After incubating in 5 wt% non-fat milk at room temperature for 1 h, the membrane was incubated with the primary antibody diluted at 1:1000 overnight at 4 °C. The membrane rinsed with TBST was incubated with the horseradish peroxidase-labeled secondary antibody at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence detection kit (Amersham Life Science, Buckinghamshire, UK). The target protein was normalized to β-actin for quantitative expression.

[0051] The above results indicate that 6-gingerol regulates H / R-induced mitophagy by inhibiting the production of mitochondrial ROS.

[0052] Example 2: 6-Gingerol alleviates PE-like mouse placental dysfunction by reducing mitophagy

[0053] To verify the effect of 6-gingerol on PE placental injury in vivo, a PE-like mouse model was established by intraperitoneal injection of L-NAME, which caused symptoms such as fetal growth restriction (FGR) and hypertension. By measuring the weights of the fetuses and placentas of each group of mice, it was found that L-NAME injection significantly reduced the weights of the fetuses and placentas in pregnant mice. In addition, compared with the L-NAME injection group, 6-gingerol increased the average weights of the fetuses and placentas, as shown in Figure 2(A). The specific experimental steps are as follows:

[0054] 10-week-old C57BL / 6 female and male mice purchased from the Experimental Animal Center of Shandong University were maintained at 18-22 °C with a 12 h / 12 h light / dark cycle and allowed free access to food and water.

[0055] Female mice were mated with male mice at a ratio of 2:1, and the time when vaginal plugs were found was recorded as gestational day 0.5. Pregnant mice were randomly divided into a CT group (n = 6), an L-NAME group (n = 6), and an L-NAME + 6-gingerol group (n = 6). Mice in the L-NAME group and the L-NAME + 6-gingerol group were subcutaneously injected with L-NAME at 125 mg / kg / d (MedChemExpress, USA) from GD9.5 to GD18.5. The L-NAME + 6-gingerol group was also intraperitoneally injected with 2 mg / kg 6-gingerol (MedChemExpress, USA) from GD8.5 to GD18.5. The CT group was intraperitoneally and subcutaneously injected with normal saline during the same period. At GD18.5, the mice were sacrificed by cervical dislocation, and the placental weight, litter size, and fetal weight were recorded. The placentas were collected and stored at -80°C or fixed with 4% paraformaldehyde for further analysis. The mitochondrial autophagy marker BNIP3 is a major protein that plays a key role in injury-induced mitochondrial autophagy. By measuring the expression of the mitochondrial autophagy marker BNIP3, immunohistochemical staining and Western blot analysis showed that the expression of BNIP3 was upregulated under the action of L-NAME, while 6-gingerol could reverse this expression. The specific experimental procedures are as follows:

[0056] At GD18.5, the mice were sacrificed by cervical dislocation, and the placentas were collected and lysed in RIPA buffer (Solarbio, Beijing, China) containing protease inhibitor (Beyotime, Beijing, China). Protein quantification was performed using a BCA kit (Solarbio, Beijing, China). Equal amounts of protein were separated on an SDS-PAGE gel and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane. After incubation in 5 wt% skim milk at room temperature for 1 hour, the membrane was incubated with the primary antibody diluted at 1:1000 overnight at 4°C. The membrane rinsed with TBST was incubated with the horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. Protein bands were visualized using an enhanced chemiluminescence detection kit (Amersham LifeScience, Buckinghamshire, UK). The target protein was normalized to β-actin for quantitative expression.

[0057] Mouse placental tissues were fixed in 4 wt% paraformaldehyde solution and embedded in paraffin. After dewaxing and rehydrating with gradient ethanol, antigen retrieval was performed on the sections. Then, the sections were blocked with goat serum (10 wt%) at room temperature for 30 min. Subsequently, the sections were incubated with the primary antibody (1:200) overnight at 4°C. After washing with PBS, the placental tissues were incubated with the secondary antibody at 37°C for 1 hour. Photographs were taken and recorded with an inverted fluorescence microscope (Olympus, BX53F, Japan); the results are shown in Figures 2(B), 2(C), and 2(D).

[0058] The above results indicate that in the placentas of preeclamptic mice, cellular mitophagy increases, leading to placental dysfunction, and 6-gingerol can reverse this process.

Claims

Application of 1.6-gingerol as a pharmaceutical ingredient in the preparation of a drug for treating preeclampsia.

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