Use of hydroxysafflor yellow A in preparation of a medicine for preventing, treating and / or relieving endometriosis

Abstract

The application provides application of hydroxysafflor yellow A in preparation of medicines for preventing, treating and / or relieving endometriosis, and belongs to the technical field of biological medicines. The application provides application of hydroxysafflor yellow A in preparation of medicines for preventing, treating and / or relieving endometriosis. Hydroxysafflor yellow A can inhibit migration and / or diffusion of endometriosis cyst, inhibit migration, invasion and abnormal planting of endometrial tissue and / or endometriosis cells outside the uterine cavity, relieve inflammatory reaction caused by endometriosis, and effectively curb lesion invasion and disease progression of endometriosis. As a main active component of safflower, hydroxysafflor yellow A provides a new direction for manufacturing new medicines for treating endometriosis by using safflower.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of hydroxysaffron yellow pigment A in the preparation of drugs for the prevention, treatment and / or relief of endometriosis. Background Technology

[0002] Endometriosis, also known as endometriosis, is a common gynecological disease in which the uterine lining (containing glands and stroma) grows outside the uterine cavity. Treatment methods for endometriosis mainly include expectant management, drug therapy, surgical treatment, and combined therapy. Apart from radical surgical treatment—removal of the entire uterus, both salpingo-oophores, and all visible lesions—other treatment methods have a high recurrence rate and cannot effectively curb lesion invasion and disease progression. Summary of the Invention

[0003] The purpose of this invention is to provide the application of hydroxysaffron yellow pigment A in the preparation of drugs for the prevention, treatment and / or relief of endometriosis, which can effectively inhibit the invasion and progression of endometriosis lesions.

[0004] This invention provides the use of hydroxysaffron yellow pigment A in the preparation of medicaments for the prevention, treatment and / or relief of endometriosis.

[0005] Preferably, the prevention, treatment, and / or relief of endometriosis includes at least one of the following: 1) Inhibits the migration and / or spread of endometriotic cysts; 2) Inhibits the migration of endometrial tissue and / or ectopic endometrial cells; 3) Inhibits the invasion of endometrial tissue and / or ectopic cells; 4) Inhibits abnormal colonization of endometrial tissue and / or ectopic cells outside the uterine cavity; 5) Relieves the inflammatory response caused by endometriosis.

[0006] Preferably, the endometriotic cells include 12Z cells.

[0007] Preferably, the inhibition of endometrial tissue and / or endometriotic cell migration is achieved by inhibiting the healing of endometriotic cell wounds.

[0008] Preferably, the prevention, treatment, and / or relief of endometriosis is achieved through at least one of the following: (1) Regulates the expression of proteins related to epithelial-mesenchymal transition and angiogenesis; (2) Reduce the expression level of inflammatory factors in translocated endometrial cells and / or inhibit the secretion of pro-inflammatory factors in translocated endometrial cells; (3) Regulate the expression of PI3K / Akt pathway-related proteins.

[0009] Preferably, the epithelial-mesenchymal transition (EMT) and angiogenesis-related proteins include E-cadherin, VEGFA, and Vimentin proteins; the regulation of EMT and angiogenesis-related protein expression includes increasing the expression level of E-cadherin protein and / or decreasing the expression levels of VEGFA and Vimentin proteins.

[0010] Preferably, the PI3K / Akt pathway-related proteins include p-PI3K and / or p-Akt.

[0011] Preferably, the regulation of PI3K / Akt pathway-related protein expression includes reducing the expression levels of p-PI3K and / or p-Akt.

[0012] Preferably, the inflammatory factors include TNF-α protein; the pro-inflammatory factors include IL-1β and / or IL-6.

[0013] Preferably, the dosage form of the drug includes an oral dosage form.

[0014] This invention provides the application of hydroxysaffron yellow pigment A in the preparation of drugs for the prevention, treatment, and / or relief of endometriosis. Hydroxysaffron yellow pigment A can inhibit the migration and / or spread of endometriotic cysts, inhibit the migration, invasion, and abnormal colonization of endometrial tissue and / or endometriotic cells outside the uterine cavity, and alleviate the inflammatory response caused by endometriosis, effectively curbing the invasion and progression of endometriosis lesions. As the main active ingredient of safflower, hydroxysaffron yellow pigment A provides a new direction for the development of novel drugs for the subsequent treatment of endometriosis using safflower. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Cell scratch map (4×); Figure 2 Cell scratch statistics; Note: Compared with the CTR group. represent p <0.01; Figure 3 Cell invasion diagram (4×); Figure 4 Cell invasion statistics; Note: Compared with the CTR group, represent p <0.01; Figure 5 Cellular E-cadherin, Vimentin, and VEGFA protein band diagram; Figure 6 Statistical graph of quantitative expression of E-cadherin, Vimentin, and VEGFA proteins in cells; Note: Compared with the CTR group. represent p <0.05; represent p <0.01; Figure 7 Cellular TNF-α protein banding diagram; Figure 8 Statistical graph of cellular TNF-α protein expression; Note: Compared with CTR, represent p <0.01; Figure 9 Statistical graph of expression levels of pro-inflammatory cytokines; Note: Compared with CTR, represent p <0.05; represent p <0.01; Figure 10 Cellular PI3K, p-PI3K, Akt, and p-Akt protein band diagram; Figure 11 Statistical graph of quantitative expression of PI3K / Akt pathway-related proteins in cells; Note: Compared with CTR. represent p <0.05; represent p <0.01; Figure 12 Bands of mouse E-cadherin, Vimentin, and VEGFA proteins; Figure 13 Statistical graphs of quantitative expression of E-cadherin, Vimentin, and VEGFA proteins in mice; Note: Compared with CTR. represent p <0.05, represent p <0.01; compared to the Model group, # represents p <0.05, ## representsp <0.01; Figure 14 Immunohistochemical images of pro-inflammatory factors IL-6 and IL-1β in mouse lesion tissue; Figure 15 Mouse TNF-α protein banding diagram; Figure 16 Statistical graph of quantitative expression of TNF-α protein in mice; Note: Compared with the CTR group. represent p <0.01; compared to the Model group, # represents p <0.05, ## represents p <<0.01; Figure 17 Bands of mouse PI3K, p-PI3K, Akt, and p-Akt proteins; Figure 18 Statistical graph of quantitative expression of PI3K / Akt pathway-related proteins in mice; Note: Compared with the CTR group. # indicates p < 0.01; compared to the Model group, # indicates p <0.05, ## represents p <0.01. Detailed Implementation

[0017] This invention provides the use of hydroxysaffron yellow pigment A in the preparation of medicaments for the prevention, treatment and / or relief of endometriosis.

[0018] In this invention, the chemical structural formula of the hydroxysaffron yellow pigment A is shown in Formula 1: Formula 1.

[0019] As one implementation method, the prevention, treatment, and / or relief of endometriosis includes at least one of the following: 1) Inhibits the migration and / or spread of endometriotic cysts; 2) Inhibits the migration of endometrial tissue and / or ectopic endometrial cells; 3) Inhibits the invasion of endometrial tissue and / or ectopic cells; 4) Inhibits abnormal colonization of endometrial tissue and / or ectopic cells outside the uterine cavity; 5) Relieves the inflammatory response caused by endometriosis.

[0020] In one embodiment, the endometriotic cells include 12Z cells.

[0021] In one implementation, the inhibition of endometrial tissue and / or endometriotic cell migration is achieved by inhibiting the healing of endometriotic cell wounds.

[0022] As one implementation method, the prevention, treatment, and / or relief of endometriosis is achieved through at least one of the following: (1) Regulation of epithelial-mesenchymal transition (EMT) and angiogenesis-related protein expression; (2) Reduce the expression level of inflammatory factors in translocated endometrial cells and / or inhibit the secretion of pro-inflammatory factors in translocated endometrial cells; (3) Regulate the expression of PI3K / Akt pathway-related proteins.

[0023] In one embodiment, the epithelial-mesenchymal transition (EMT) and angiogenesis-related proteins include E-cadherin, VEGFA, and Vimentin proteins; the regulation of E-cadherin and angiogenesis-related protein expression includes increasing the expression level of E-cadherin protein and / or decreasing the expression levels of VEGFA and Vimentin proteins.

[0024] In one implementation, the PI3K / Akt pathway-related proteins include p-PI3K and / or p-Akt.

[0025] As one implementation, the regulation of PI3K / Akt pathway-related protein expression includes reducing the expression levels of p-PI3K and / or p-Akt.

[0026] In one embodiment, the inflammatory factors include TNF-α protein; the pro-inflammatory factors include IL-1β and / or IL-6.

[0027] In one embodiment, the hydroxysaffron yellow pigment A includes saffron yellow pigment for injection; the saffron yellow pigment for injection is commercially available. In the specific implementation of this invention, the saffron yellow pigment for injection is purchased from Zhejiang Yongning Pharmaceutical Co., Ltd., named Letan® saffron yellow pigment for injection, with a specification of 50mg per vial (containing 42.5mg of hydroxysaffron yellow pigment A); the mass percentage of hydroxysaffron yellow pigment A in the saffron yellow pigment for injection is 85%, with the remainder being the excipient mannitol.

[0028] As one implementation method, the dosage form of the drug includes an oral dosage form.

[0029] In one embodiment, the drug is a unit-dose formulation; based on mice, the unit-dose formulation is prepared to facilitate administration at a dose of 25-100 mg / kg, and further, the unit-dose formulation is prepared to facilitate administration at a dose of 50-100 mg / kg.

[0030] To further illustrate the present invention, the application of hydroxysaffron yellow A provided by the present invention in the preparation of medicaments for the prevention, treatment and / or relief of endometriosis is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0031] The experimental materials used in the following experimental examples include: 12Z cells (Shanghai Fuheng Biotechnology Co., Ltd.); Safflower Yellow for Injection (Zhejiang Yongning Pharmaceutical Co., Ltd., 50mg per vial, containing 42.5mg of hydroxysafflower yellow A). Fetal bovine serum (Gibco); F12 medium (Gibco); BALB / c female mice (Shihezi Helilai Laboratory Equipment Sales Department); BCA protein concentration assay kit (Hefei Baisha Biotechnology Co., Ltd.); Western blot primary antibodies, TNF-α, E-daherin, ER, PR (Shenyang Wanlei Biotechnology Co., Ltd.); Vimentin, VEGFA (Beijing Bio-Sens Biotechnology Co., Ltd.); PI3K, β-actin, GAPDH (Wuhan Sanying Biotechnology Co., Ltd.); p-PI3K (Beinlai Biotechnology Co., Ltd.), Akt, p-Akt (Aibimat Biopharmaceutical (Shanghai) Co., Ltd.); ECL luminescence assay kit (Wuhan Sanying Biotechnology Co., Ltd.); IL-6, IL-1β enzyme-linked immunosorbent assay kit (Beinlai Biotechnology Co., Ltd.).

[0032] Experimental Example 1: Effect of hydroxysaffron yellow A on the migration and invasion of 12Z cells 1. Experimental Methods 1.1 Scratch assay to detect cell migration Take a sterile 6-well plate and draw three evenly spaced horizontal lines on the back of each well as photographic markers. Take 12Z cells in the logarithmic growth phase, digest them with trypsin, resuspend them in complete culture medium, and count them. Divide the plate at 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 g / mL in 6-well plates. The plates were then incubated overnight at 37°C with 5% CO2. Once the cells reached confluence of 80% or more, the plates were removed from the incubator. Using a 200 μL sterile pipette tip, straight lines were drawn on the cells along the diameter of the wells, perpendicular to the bottom horizontal line. After drawing the lines, the culture medium in the wells was carefully aspirated, and PBS was slowly added along the well walls to gently wash the cells 2-3 times to thoroughly remove cell debris. After washing, serum-free culture medium containing different doses of the drug was added to each well (0.1, 0.2, and 0.4 g / mL for low, medium, and high doses of hydroxysaffron yellow A (SL, SM, and SH groups, respectively; 25 μM for the Dienogest group). After drug administration, the cells were observed under an inverted microscope, and photographs were taken at three locations (top, middle, and bottom) on each line, and the result was recorded as 0 h. The 6-well plates were returned to the incubator and incubated for 48 hours. Images were acquired again after 48 hours, and the result was recorded. The images were analyzed using ImageJ, and the cell scratch healing rate (%) was calculated as: (0-hour scratch area - 48-hour scratch area) / 0-hour scratch area × 100%. 1.2 Transwell assay for detecting invasion Matrigel was thawed overnight at 4°C on ice, and then diluted 1:8 with pre-chilled medium. The required Transwell chambers were placed in 24-well plates. 50 μL of the diluted Matrigel was evenly added to the surface of the microporous membrane in the upper chamber of each Transwell. The plates were then incubated at 37°C for 2 hours to allow the Matrigel to polymerize on the membrane. The plates were removed from the incubator, and 100 μL of serum-free medium was added to the upper chamber. The plates were then incubated at room temperature for 30 minutes to hydrate the basement membrane. Simultaneously, 12Z cells in the logarithmic growth phase were digested with trypsin, resuspended in serum-free drug-containing medium, and the cell density adjusted to 6 × 10⁶ cells / well. 4Cells / mL. After hydration, the culture medium in the upper chamber was aspirated, and 100 μL of cell suspension was added to the upper chamber of the Transwell. 500 μL of complete culture medium was added to the lower chamber. The 24-well plate was incubated at 37°C with 5% CO2 for 48 h. After incubation, the Transwell chamber was carefully removed, the liquid in both chambers was removed, and the cells were washed twice with PBS. Uninvaded cells and the Matrigel layer on the inner surface of the upper chamber membrane were gently wiped away with a moistened sterile cotton swab. The chamber was transferred to a well containing 4% paraformaldehyde solution and fixed at room temperature for 20 min. After rinsing twice with PBS, the chamber was then placed in 0.1% crystal violet staining solution and stained at room temperature in the dark for 15 min. After staining, the chamber was repeatedly and gently rinsed with deionized water to remove background dye, and then air-dried. After the membrane was completely dry, at least five different fields of view were randomly selected under an inverted microscope for photographing and counting. The images were processed using ImageJ, and the cell invasion rate (%) was calculated as: (number of cells in the drug treatment group / number of cells in the control group) × 100%.

[0033] 1.3 Western Blot analysis of expression levels of migration and invasion proteins After treating cells in the logarithmic growth phase according to the experimental design for 48 hours, the culture medium was discarded, and the cells were washed twice with pre-chilled PBS. An appropriate amount of pre-chilled RIPA lysis buffer (containing a mixture of 1 mM PMSF and protease inhibitors) was added to each well, and cells were lysed on ice for 30 min. Lysed cells were collected and centrifuged at 4°C and 12000 rpm for 15 min, and the supernatant was collected. Protein concentration was determined using the BCA method. Protein samples were mixed with 4× loading buffer and boiled in water for 10 min to ensure complete protein denaturation. A 10% separating gel and stacking gel were prepared. Samples and markers were added sequentially to the loading wells. Electrophoresis was performed in the electrophoresis buffer, first at a constant voltage of 60 V to pass the sample through the stacking gel, then switched to a constant voltage of 120 V for separation until the bromophenol blue indicator line reached the bottom of the gel. After electrophoresis, the gel, PVDF membrane (pre-activated with methanol), filter paper, and sponge pad were assembled in a "sandwich" structure in the transfer buffer. Transfer was performed at a constant current of 300 mA for 90 min under ice bath conditions. After transfer, the PVDF membrane was removed and placed in TBST blocking buffer containing 5% skim milk, and blocked on a shaker at room temperature for 2 hours. The membrane was washed with TBST on a shaker (4 times, 5 min each time). Protein-specific primary antibody was added and incubated overnight at 4 °C. The primary antibody was recovered, and the membrane was washed again. The membrane was then incubated with the corresponding secondary antibody on a shaker at room temperature for 1 hour. After incubation, the membrane was washed again. Equal volumes of ECL chemiluminescence reagent A and B were mixed and uniformly added to the membrane. The signal image was captured using a chemiluminescence imaging system. The gray values ​​of the target protein and internal control protein bands were measured using ImageJ, and statistical analysis was performed.

[0034] 2. Experimental Results 2.1 Hydroxysafflower yellow pigment A inhibits 12Z cell migration The wound healing of 12Z cells after 48 hours of treatment with hydroxysaffron yellow A and dinogest was assessed using a scratch assay. Figure 1 and Figure 2 As shown, hydroxysaffron yellow A can inhibit cell wound healing in a concentration-dependent manner, and compared with the control group, the cell scratch healing rate decreased by 13%, 19%, and 23%, respectively. p <0.01); the dinogest group also saw a 24% reduction ( p <0.01), with no significant difference from hydroxysaffron yellow A in any group.

[0035] 2.2 Hydroxysafflower yellow pigment A inhibits 12Z cell invasion The invasive ability of 12Z cells was observed using Transwell assays. Figure 3 and Figure 4 As shown, after 48 h of treatment with different concentrations of the drug on 12Z cells, compared with the control group, the number of invasive cells gradually decreased with the increase of hydroxysaffron yellow A concentration, indicating that hydroxysaffron yellow A can significantly inhibit the invasive ability of 12Z cells. p <0.01), and showed a dose-dependent relationship; there was no significant difference between the dinogest group and the high-dose group.

[0036] Effects of 2,3-hydroxysafflower yellow pigment A on 12Z cell migration and invasion-related proteins Western blot analysis was used to detect changes in the expression of epithelial-mesenchymal transition (EMT) and angiogenesis-related proteins in cells after 48 h of treatment with hydroxysaffron yellow A. Results are as follows: Figure 5 and Figure 6 As shown, compared with the control group, the expression level of E-cadherin protein in 12Z cells treated with hydroxysaffron yellow A was increased, while the expression levels of VEGFA and Vimentin proteins were decreased, both in a concentration-dependent manner. There was no significant difference between the dinogest group and the hydroxysaffron yellow A group. In conclusion, hydroxysaffron yellow A can inhibit 12Z migration and invasion.

[0037] Experimental Example: Effect of 2-hydroxysafflower yellow pigment A on inflammatory factors in 12Z cells 1. Experimental Methods 1.1 Western Blot analysis of TNF-α expression level (a 12Z inflammatory cytokine) Following the same procedure as in Example 1, step 1.3, the expression level of TNF-α protein was detected.

[0038] 1.2 ELISA detection of pro-inflammatory factors IL-1β and IL-6 The cell supernatant culture medium was collected after 48 hours of treatment, and the cytokine levels of IL-6 and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.

[0039] 2. Experimental Results 2.1 Effects of hydroxysaffron yellow pigment A on TNF-α protein in 12Z cells The expression level of TNF-α protein in 12Z cells after treatment with hydroxysaffron yellow A for 48 h is as follows: Figure 7 and Figure 8 As shown, hydroxysaffron yellow pigment A reduced the expression level of TNF-α protein in a concentration-dependent manner, with significant reductions observed in the medium and high dose groups. p <0.01); Although the dinogest group could significantly reduce protein expression levels, the effect was not as good as the medium and high dose groups of hydroxysaffron yellow pigment A.

[0040] Effects of 2-hydroxysafflower yellow pigment A on the secretion of pro-inflammatory factors by 12Z cells The levels of pro-inflammatory cytokines were detected using enzyme-linked immunosorbent assay (ELISA). Results are as follows: Figure 9 As shown, after treating cells with different concentrations of hydroxysaffron yellow pigment A for 48 hours, the secreted pro-inflammatory factors IL-1β and IL-6 decreased in a concentration-dependent manner. The effect of the dinogest group was not as good as that of the medium and high dose groups of hydroxysaffron yellow pigment A. The results indicate that hydroxysaffron yellow pigment A can reduce the level of pro-inflammatory factors in 12Z cells.

[0041] Experimental Example 3: Effects of hydroxysafflower yellow pigment A on PI3K / Akt pathway-related proteins in 12Z cells 1. Experimental Methods Following the same procedure as in Example 1, step 1.3, the expression levels of the key proteins PI3K, p-PI3K, Akt, and p-Akt in the PI3K / Akt pathway were detected.

[0042] 2. Experimental Results The regulatory effect of hydroxysaffron yellow pigment A on the PI3K / Akt signaling pathway in 12Z cells was investigated by Western blot. The results are as follows: Figure 10 and Figure 11 As shown, hydroxysaffron yellow A significantly downregulated the expression of p-PI3K and p-Akt in 12Z cells, while the expression levels of PI3K and Akt did not change significantly. Therefore, hydroxysaffron yellow A may exert its pharmacological effects by downregulating the key proteins p-PI3K and p-Akt in the PI3K / Akt signaling pathway, thereby inhibiting the activation of this pathway.

[0043] Experiment 4: Effects of hydroxysafflower yellow pigment A on mice with endometriosis 1. Experimental Methods Female BALB / c mice aged 6-8 weeks were selected and acclimatized for one week. Estradiol was injected intraperitoneally on days 1 and 4 to unify the estrous cycle. Allogeneic transplantation was performed via intraperitoneal injection. The donor mice were dissected, and the uterus was removed and cleaned of blood and mucus with pre-cooled saline. The uterine horn was bisected and placed in 0.5 mL of pre-cooled saline. The uterus was longitudinally dissected using ophthalmic scissors to expose the endometrial layer, which was then cut into pieces ≤1 mm in volume. 3 The endometrial fragments were prepared into a suspension for later use. Recipient mice were anesthetized, and a 20 mL syringe needle was attached to a 1 mL syringe barrel. The needle was inserted approximately 0.5 cm to the left of the midline of the lower abdomen of the mouse, and the suspension was injected. After injection, the injection site was immediately pressed with a cotton swab for 30 seconds to prevent leakage of endometrial fragments. One donor mouse was used to inject two recipient mice. On postoperative days 1 and 4, estradiol solution was injected subcutaneously to promote lesion development.

[0044] A mouse model was established. On day 7 after modeling, the model mice were divided into four groups: a low-dose hydroxysaffloryellow A group (SL group), a medium-dose hydroxysaffloryellow A group (SM group), a high-dose hydroxysaffloryellow A group (SH group), a positive control dienogest group (Dienogest group), a model group, and a control group (Control group). Mice in each group were administered the drug via gavage for 21 consecutive days. The dosage was 1 mL / 100g (1 mL per 100g of mouse body weight). The specific daily administration regimen and dosage were as follows: the Dienogest group received dienogest 0.2 mg / kg; the hydroxysaffloryellow A groups were administered hydroxysaffloryellow A as follows: SL group 25 mg / kg, SM group 50 mg / kg, and SH group 100 mg / kg. The Model and Control groups received saline. On day 28 after modeling, mice were euthanized and dissected. Ectopic endometrial tissue was fixed and immunohistochemically analyzed for changes in the expression of inflammation-related proteins. Uterine tissue was collected and Western blot was used to detect the levels of migration and invasion, inflammatory factors, and PI3K / Akt pathway-related proteins in each group of mice.

[0045] 2. Experimental Results 2.1 Hydroxysafflower yellow A inhibits the expression of migration-invasion related proteins in mice. The expression of E-cadherin, Vimentin, and VEGFA proteins in the uterine tissues of mice in each group was detected by Western blotting. The results are as follows: Figure 12 and Figure 13 As shown, compared with the control group, the E-cadherin protein level in the model group was significantly reduced ( p <0.05), the expression levels of VEGFA and Vimentin proteins were significantly increased (p <0.01). Compared with the model group, the expression level of E-cadherin protein in mice in the hydroxysaffron yellow A group increased in a concentration-dependent manner, with the high-dose group showing a significant increase ( ). p <0.05), there was no significant difference between the hydroxysaffron yellow pigment A group and the dinogest group; however, the expression level of VEGFA decreased in a concentration-dependent manner, with significant differences observed in the medium and high dose groups. p <0.01), with no significant difference from the dinogest group; with increasing drug concentration, the expression level of Vimentin gradually decreased (in a concentration-dependent manner), with significant differences observed in the high-dose group. p <0.05), there was no significant difference between each group and the dinogest group.

[0046] 2,2-Hydroxysafflower yellow pigment A inhibits the expression of inflammatory factors in mice. Immunohistochemical staining results as follows Figure 14 The results showed that IL-6 and IL-1β positive signals (brownish-yellow granules) were mainly located in the cytoplasm of endometrial epithelial cells and stromal cells. In the model group, IL-6 and IL-1β proteins were highly expressed in the endometrial tissue, with strong positive staining intensity, a large number of positive cells, and a diffuse distribution. Numerous inflammatory cells were densely infiltrated in the stroma, indicating an active inflammatory response in the endometrium of the model group. Compared with the model group, the hydroxysaffron yellow pigment A group showed significantly weaker IL-6 and IL-1β positive staining intensity, a significantly reduced brownish-yellow signal, a significantly lower number of positive cells, and sparser distribution. Inflammatory cell infiltration in the stroma was significantly reduced, suggesting that the drug effectively inhibited the expression of IL-6 and IL-1β proteins in ectopic endometrial tissue and significantly alleviated the local inflammatory response. The effect of the dinogest group was not significantly different from that of the drug-treated group.

[0047] The expression of TNF-α in the uterine tissues of mice in each group was detected by Western blotting, and the results are as follows: Figure 15 and Figure 16 As shown, compared with the control group, the model histone levels were significantly increased ( p <0.01); compared with the model group, hydroxysaffron yellow pigment A significantly reduced the protein level of TNF-α; there was no significant difference between the dinogest group and the medium and high dose groups of hydroxysaffron yellow pigment A.

[0048] 2,3-hydroxysafflower yellow pigment A exerts its therapeutic effect on endometriosis in vivo through the PI3K / Akt pathway. The expression of PI3K / Akt signaling pathway-related proteins in the uterine tissues of mice in each group was detected by Western blot. The results are as follows: Figure 17 and Figure 18As shown, compared with the control group, the levels of p-PI3K and p-Akt proteins in the model group were significantly increased ( p <0.01); Compared with the model group, hydroxysaffron yellow A significantly downregulated the expression of p-PI3K and p-Akt, while the expression levels of PI3K and Akt did not change significantly. Therefore, hydroxysaffron yellow A may exert a therapeutic effect on endometriosis by downregulating the key proteins p-PI3K and p-Akt in the PI3K / Akt signaling pathway, thereby inhibiting the activation of this pathway.

[0049] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of hydroxysaffron yellow pigment A in the preparation of drugs for the prevention, treatment and / or relief of endometriosis.

2. The application according to claim 1, characterized in that, The prevention, treatment, and / or relief of endometriosis includes at least one of the following: 1) Inhibits the migration and / or spread of endometriotic cysts; 2) Inhibits the migration of endometrial tissue and / or ectopic endometrial cells; 3) Inhibits the invasion of endometrial tissue and / or ectopic cells; 4) Inhibits abnormal colonization of endometrial tissue and / or ectopic cells outside the uterine cavity; 5) Relieves the inflammatory response caused by endometriosis.

3. The application according to claim 2, characterized in that, The endometriotic cells include 12Z cells.

4. The application according to claim 2, characterized in that, The inhibition of endometrial tissue and / or endometriotic cell migration is achieved by inhibiting the healing of endometriotic cell wounds.

5. The application according to claim 1, characterized in that, The prevention, treatment and / or relief of endometriosis are achieved through at least one of the following: (1) Regulates the expression of proteins related to epithelial-mesenchymal transition and angiogenesis; (2) Reduce the expression level of inflammatory factors in translocated endometrial cells and / or inhibit the secretion of pro-inflammatory factors in translocated endometrial cells; (3) Regulate the expression of PI3K / Akt pathway-related proteins.

6. The application according to claim 5, characterized in that, The epithelial-mesenchymal transition (EMT) and angiogenesis-related proteins include E-cadherin, VEGFA, and Vimentin proteins; the regulation of EMT and angiogenesis-related protein expression includes increasing the expression level of E-cadherin protein and / or decreasing the expression levels of VEGFA and Vimentin proteins.

7. The application according to claim 5, characterized in that, The PI3K / Akt pathway-related proteins include p-PI3K and / or p-Akt.

8. The application according to claim 5, characterized in that, The regulation of PI3K / Akt pathway-related protein expression includes reducing the expression levels of p-PI3K and / or p-Akt.

9. The application according to claim 5, characterized in that, The inflammatory factors include TNF-α protein; the pro-inflammatory factors include IL-1β and / or IL-6.

10. The application according to claim 1, characterized in that, The dosage form of the drug includes oral dosage forms.

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