Application of hesperetin in preparation of medicine for treating intrauterine adhesion

Abstract

The invention provides application of hesperetin in preparation of a medicine for treating intrauterine adhesion, and belongs to the technical field of endometrial repair. It is found that hesperetin can promote endometrial regeneration, inhibit endometrial fibrosis and promote endometrial tissue angiogenesis and can be used for preparing the medicine for preventing and / or treating intrauterine adhesion. And hesperetin is a natural flavone compound, exists in various citrus fruits, has excellent safety and a wide source, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of endometrial repair, and particularly relates to application of hesperetin in preparation of a medicine for treating intrauterine adhesion. BACKGROUND

[0002] Intrauterine adhesion (IUA), also known as Asherman syndrome, is mainly caused by traumatic factors such as intrauterine surgery and infection, and is an important cause of female infertility, recurrent miscarriage and menstrual abnormalities. The core pathological change is that after the basal layer of the endometrium is damaged, the repair process is unbalanced, which is manifested as abnormal or excessive fibrosis and regeneration disorders of endometrial epithelial and stromal cells, and finally forms scar tissue adhesion.

[0003] At present, the standard treatment scheme for IUA in the clinic is to perform adhesion separation under hysteroscopy, and postoperative hormone treatment such as estrogen is used to promote endometrial repair. However, this strategy has significant limitations: the surgery itself can cause secondary trauma, and the effect of hormone treatment on inhibiting fibrosis is limited, resulting in a high recurrence rate and unsatisfactory overall efficacy. Therefore, developing a new drug that can simultaneously target to promote functional regeneration of the endometrium (including epithelial repair and vascular reconstruction) and effectively inhibit the fibrosis process has become a key problem to be solved in the clinic. SUMMARY

[0004] In order to solve the problems in the prior art, the first purpose of the present application is to provide application of hesperetin in preparation of a medicine for preventing and / or treating intrauterine adhesion, which can promote endometrial regeneration, inhibit endometrial fibrosis and promote endometrial tissue angiogenesis.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides application of hesperetin in preparation of a medicine for treating intrauterine adhesion.

[0006] The present application also provides application of hesperetin in preparation of a medicine for preventing intrauterine adhesion.

[0007] The present application also provides application of hesperetin in preparation of a medicine for promoting endometrial regeneration.

[0008] The present application also provides application of hesperetin in preparation of a medicine for inhibiting endometrial fibrosis.

[0009] The present application also provides application of hesperetin in preparation of a medicine for promoting endometrial tissue angiogenesis.

[0010] Preferably, the hesperetin inhibits collagen deposition and expression of alpha-SMA.

[0011] Preferably, the hesperidin upregulates Hoxa10 and progesterone receptors, improving endometrial receptivity.

[0012] Preferably, the hesperidin promotes the expression of CD31 and KI67.

[0013] Preferably, the effective concentration of hesperidin is 3~8 μM.

[0014] Preferably, the drug further includes pharmaceutically feasible excipients.

[0015] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention is the first to discover that hesperidin has a dual mechanism of action: hesperidin can simultaneously target and promote the proliferation of endometrial cells (such as stromal cells) and angiogenesis of vascular endothelial cells, and effectively inhibit the expression of key markers of fibrosis (such as α-SMA and COL1A1), thus synergistically repairing the endometrium from two dimensions of regeneration and anti-fibrosis, solving the problem of current therapies neglecting one aspect.

[0016] This invention reveals that hesperidin is closely related to the inhibition of TGFβ-Smad, a core signaling pathway in fibrosis, with a clearly defined target. In vivo animal experiments have confirmed that hesperidin can effectively restore endometrial thickness in IUA model mice, promote microvascular formation, and significantly reduce the fibrotic area, demonstrating superior efficacy compared to current conventional therapies. Furthermore, the hesperidin described in this invention has a low recurrence rate. By fundamentally regulating the fibrosis process, it can significantly reduce the recurrence rate after intrauterine adhesion surgery, while also possessing advantages such as high efficiency, low cost, low risk, and high success rate, showing broad application prospects. Attached Figure Description

[0017] Figure 1 Drug screening and validation of fibrosis inhibition effects; Figure 2 Hesperidin has effects on the proliferation and migration of endometrial stromal cells and on the formation of tubes in human vascular endothelial cells. Figure 3 The results showed that hesperidin promoted regeneration and inhibited fibrosis in mice; Figure 4 Hesperidin promotes angiogenesis and cell proliferation in mice. Figure 5 Hesperidin improves endometrial receptivity. Figure 6 Safety evaluation of in situ administration of hesperidin; Figure 7 Hesperidin inhibits fibrosis and promotes regeneration in human endometrial organoids. Detailed Implementation

[0018] This invention provides the application of hesperidin in the preparation of drugs for preventing and / or treating intrauterine adhesions, drugs for promoting endometrial regeneration, drugs for inhibiting endometrial fibrosis, and drugs for promoting angiogenesis in endometrial tissue. The hesperidin can be effectively extracted from citrus fruits such as oranges and tangerines, or is commercially available through conventional means.

[0019] The core of intrauterine adhesions (IUA) is endometrial hypoplasia and fibrosis. When the basal layer of the endometrium is damaged, the regenerative capacity of stromal cells is disrupted or depleted. The repair process transforms into abnormal repair dominated by fibroblasts and myofibrils, leading to massive collagen deposition, scar tissue formation, and a lack of normal glands and blood vessels, ultimately resulting in adhesions. This invention discovered that hesperidin is closely related to the inhibition of TGFβ-Smad, a core fibrosis signaling pathway, and verified that hesperidin inhibits collagen deposition and α-SMA expression, promotes CD31 and KI67 expression, and exhibits excellent effects in promoting endometrial repair and anti-fibrosis in both in vitro and in vivo models. It demonstrates that hesperidin can restore endometrial thickness in patients with intrauterine adhesions, promote endometrial angiogenesis and endometrial cell regeneration, and also has the ability to inhibit fibrosis and promote proliferation at the level of complex human endometrial organoids.

[0020] The expression of genes related to endometrial receptivity is an important indicator for evaluating the recovery of uterine function. IUA patients have reduced expression of receptivity-related genes. The hesperidin described in this invention can effectively upregulate two indicators, Hoxa10 and progesterone receptor (PR), proving that the hesperidin can effectively improve endometrial receptivity.

[0021] The effective concentration of hesperidin described in this invention is 3~8μM, more preferably 4μM, 5μM, 6μM or 7μM.

[0022] The drug of the present invention may use hesperidin as the sole active ingredient, and the drug may also include pharmaceutically feasible excipients.

[0023] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0024] In a specific embodiment of the present invention, the hesperidin used was purchased from Stelleck Chemicals (Houston, America), product batch number S2308.

[0025] Unless otherwise specified, the following embodiments are all conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1 Six candidate drugs, including Enzastaurin, Hesperetin, Pirfenidone, Ruboxistaurin, Staurosporine, and Valrubicin, were pre-selected from the FDA-approved drug library L1300 based on their ability to inhibit TGF-β and were evaluated.

[0028] Three concentration gradients were set for each drug: Enzastaurin (0.1μM, 1μM, 10μM), Hesperetin (1μM, 5μM, 50μM), Pirfenidone (1μM, 10μM, 50μM), Ruboxistaurin (0.1μM, 1μM, 10μM), Staurosporine (0.01μM, 0.1μM, 1μM), and Valrubicin (0.1μM, 1μM, 10μM). Each drug group, combined with TGF-β1, was cultured with endometrial stromal cells (ESCs) for 5 days. An NC group (blank control group, no drugs added) and a TGF-β1-only group (positive control group) were set up. Toxic drug concentrations were screened using CCK-8 assay (Yeasen, Shanghai, China). Solarbio G1473 was used to quantify collagen accumulation using Sirius red staining, OD... 540 To quantify collagen quantity and eliminate the influence of cell count on the results, OD was used. 540 / OD 450 The proportion of drugs that inhibit the fibrosis process and their appropriate concentrations were screened.

[0029] TGFβ1 is a pro-fibrotic factor and was used as a positive control group. The positive control group consisted of 10 ng / ml TGFβ1 and endometrial stromal cells (ESCs) cultured together. The experimental groups consisted of 10 ng / ml TGFβ1 supplemented with 1 μM and 5 μM hesperidin, respectively. After 72 h of culture, COL1A1 and ACTA2 fluorescence were detected using Beyotime CST72026S and AF0048, and Sirius red staining was performed. The expression levels of COL1A1 and ACTA2 were detected using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan) and SYBR Green Fast qPCR kit mix (Abclone, Wuhan, China).

[0030] Drug screening and validation of fibrosis inhibition effects, such as Figure 1 As shown in the figure, A represents the drug screening process, B represents the inhibitory effect of six drugs on fibrosis, C represents the fluorescence detection of COL1A1 and ACTA2 in inhibiting fibrosis with hesperidin, D represents the relative fluorescence intensity of COL1A1 and ACTA2, E represents the Sirius red staining results of hesperidin inhibiting fibrosis, and F represents the effect of hesperidin on the expression levels of COL1A1 and ACTA2. The results show that hesperidin was the optimal drug selected through screening, and 5 μM was found to be its optimal concentration for in vitro action. TGFβ1 stimulation significantly upregulated collagen deposition and the expression of COL1A1 and ACTA2, and 5 μM hesperidin could inhibit this effect. These findings indicate that hesperidin can serve as a safe and potent fibrosis inhibitor with significant efficacy at a concentration of 5 μM in vitro.

[0031] Example 2 Experimental Procedure: Human endometrial stromal cells (HESCs) were cultured with a final concentration of 5 μM hesperidin, and a blank control group (NC group, without any added drug) was set up. To investigate the effect of hesperidin on the proliferation of HESCs: CCK8 assays were performed on days 0, 1, 3, and 5 of culture, and OD values ​​were recorded. 450 To investigate the effect of hesperidin on the migration ability of HESCs: scratch experiments were performed on HESCs at 0h, 6h, 12h, and 24h of culture, and the migration rate was counted. The transcriptome level and protein expression level of maker KI67, which is related to proliferation, were detected at 48h and 72h of culture. To investigate the angiogenesis effect of hesperidin on human umbilical vein endothelial cells (HUVECs), an NC group (blank control group, without any drugs) was set up: images of HESCs were taken at 2h, 4h, 6h, and 8h to capture tube formation images of endothelial cells, and the number of connections, meshes, and trunks was calculated. The total branch length was measured to quantify the functional changes of endothelial cells.

[0032] Hesperidin's effects on endometrial stromal cell proliferation and migration, and its effects on human vascular endothelial cell tube formation are as follows: Figure 2 As shown in the figure, a represents the proliferation of HESCs in the two groups on days 0, 1, 3, and 5, and b represents the OD. 450Statistical analysis was performed, with c representing representative images of HESCs migration at 0, 6, 12, and 24 hours after treatment in both groups; d representing cell migration rate statistics; e representing KI67 immunofluorescence staining of HESCs in both groups at 72 hours of culture; f representing the transcriptomic level of maker KI67 in HESCs in both groups at 48 hours of culture; g representing the protein expression level of maker KI67 in HESCs in both groups at 72 hours of culture; h representing representative images of in vitro HUVECs vascular network formation in both groups; and i representing the statistical results of connection number, mesh number, trunk number, and total branch length. The results showed that the addition of 5 μM hesperidin promoted the proliferation and migration of human endometrial stromal cells, directly indicating that it may enhance the self-repair capacity of the endometrium. Compared with the blank control group, the proliferation-related maker... KI67 It promotes proliferation at both the transcriptomic and protein expression levels. At 8 h of induction of HUVECs, compared with the blank control group, the hesperidin group showed an increase in the number of connections, meshes, trunks, and total branch length.

[0033] Example 3 The IUA mouse model was created through a combination of mechanical injury and infection, as follows: Laboratory mice were randomly divided into three groups: sham-operated group, IUA group, and treatment group. In the IUA group, a small incision was made 0.5 cm above the cervix on the modeling side. The endometrium was turned outward 1 cm along the incision using forceps, and after tearing off the endometrium, the normal shape of the uterus was restored. 20 μl of 10 mg / L LPS was injected into the lesion site to induce infection. The incision was sutured with 7-0 sutures, and the start and end points of the modeling were marked with sutures. The other side of the uterus without modeling was left untreated. In the treatment group, the modeling side of the uterus was supplemented with 5 μM hesperidin in the LPS solution, similar to the IUA group. The other side of the uterus without modeling was left untreated. In the sham-operated group, the endometrium of the modeling side of the uterus was turned outward and exposed for 20 minutes, followed by an injection of only 20 μl of PBS. Tissue samples were collected two weeks after the operation.

[0034] The samples from each group were compared in appearance and stained with hematoxylin and eosin (HE). The endometrial thickness of the three groups of mice was statistically analyzed, and Masson staining was used to assess the distribution of collagen fibers in the uterine sections. Since the animal experiment used unilateral modeling, O represents the modeled uterus and N represents the non-modeled uterus. To exclude individual differences among mice, the O / N ratio was used to compare the endometrial condition of the three groups.

[0035] The results of hesperidin promoting regeneration and inhibiting fibrosis in mice are as follows: Figure 3As shown in the figure, a represents the experimental procedure, b represents the comparison of samples from both sides of the uterus in each group of mice, c represents HE staining of the uterus in each group of mice, d represents the endometrial thickness statistics, e represents Masson staining of the uterus in each group of mice, and f represents the percentage of fibrosis area. The results showed that compared to the sham group, the uterus at the surgical site in the IUA group was significantly congested and swollen. Compared to the undamaged side of the uterus, the endometrium in the IUA group tended to shrink and thin. The modeled side of the uterus in the treatment group was translucent and shiny, similar to the normal uterus on the undamaged side. Compared to the sham group and the treatment group, the endometrial thickness on the modeled side of the IUA group was significantly thinner, while there was no significant difference between the treatment group and the sham group (O / N 0.95±0.07% in the sham group, 0.62±0.06% in the IUA group, and 1.00±0.11% in the treatment group), indicating that hesperidin can restore the endometrial thickness in mice with intrauterine adhesions. Endometrial collagen fibers appeared blue, and damaged endometrial tissue showed a deep blue staining, suggesting excessive collagen deposition. After hesperidin intervention, the staining intensity decreased significantly, reflecting a reduction in collagen accumulation. The endometrial staining in the treatment group was very similar to that in the sham surgery group. The relative fibrosis area (O / N) quantitative data are as follows: sham surgery group 0.98±0.12%, IUA model group 5.43±0.68%, treatment group 1.75±0.29%.

[0036] CD31 immunohistochemical staining was performed on the three groups of uterus tissues, and the ratio of CD31 positive area was calculated. The expression of KI67, a marker of regeneration, in the uterine tissues of the three groups was detected by immunohistochemistry, and the ratio of KI67 positive area was calculated.

[0037] Hesperidin promotes angiogenesis and cell proliferation in mice, as shown in the following results. Figure 4 As shown in the figure, a) is CD31 immunohistochemical staining, b) is KI67 immunohistochemical staining, c) is the CD31 positive area ratio, and d) is the KI67 positive area ratio. The results showed that the relative positive O / N ratio for CD31 in the IUA group was 0.1042±0.0386%. After hesperidin treatment, this ratio increased to 1.161±0.358%, similar to the 1.243±0.261% in the sham-operated group. This demonstrates that hesperidin can promote endothelial angiogenesis in IUA mice. The relative positive O / N ratio for KI67 in the IUA group was 0.3605±0.0542%. After hesperidin treatment, this ratio increased to 1.031±0.153%, similar to the 0.927±0.086% in the sham-operated group. This demonstrates that hesperidin can promote endothelial cell regeneration in IUA mice.

[0038] Immunofluorescence staining and immunohistochemistry were used to quantify two indicators, HOXA10 and progesterone receptor (PR), to assess whether hesperidin improves endometrial receptivity.

[0039] Hesperidin improves endometrial receptivity as shown by Figure 5 As shown in the figure, a represents the immunofluorescence staining results, b represents the immunohistochemical staining results, c represents the statistical results of the HOXA10 index, and d represents the statistical results of the PR index. The results showed that the expression of HOXA10 and PR was significantly upregulated after hesperidin treatment (0.971±0.040%, 0.847±0.136%, respectively), which was statistically significant compared with the IUA group (0.048±0.009%, 0.328±0.107%), but not significantly different from the sham-operated group (0.963±0.063%, 0.977±0.100%).

[0040] To assess the safety of in situ administration of hesperidin, heart, liver, spleen, lungs, and kidneys were collected from mice on day 14 for hematoxylin-eosin (H&E) staining. Biochemical indicators related to these major organs were also measured. Results are as follows: Figure 6 As shown in the figure, a represents the hematoxylin-eosin (H&E) staining of important organs in each group, and b represents the biochemical indicators of important organs in each group. The results showed no significant differences among the three groups, indicating that liver, kidney, and heart functions and overall metabolic status remained normal. This suggests that hesperidin has good stability and safety in vivo and is a safe drug for treating intrauterine adhesions.

[0041] Example 4 Human endometrial organoids were constructed by assembling stromal cells into epithelial cells. Immunofluorescence staining of epithelial and stromal cell markers confirmed successful model construction. The construction method is as follows: Endometrial epithelial cell organoids in gel droplets were digested with Tryple solution in a 37°C water bath for 15 minutes. After digestion was terminated, the cells were centrifuged (500g, 5 minutes) to obtain an epithelial cell pellet. Simultaneously, endometrial stromal cells expanded to 80-90% were digested with trypsin solution and centrifuged (200g, 5 minutes) to obtain a stromal cell pellet. Epithelial cells and stromal cells were resuspended separately and counted. Then, the cells were analyzed at 5 × 10⁻⁶ cells / mL. 4 Resuspensions of stromal cells and epithelial cells were prepared at a cell concentration of / ml (1:1 ratio). After mixing, 2% of pre-thawed (4°C) matrix gel was added and the mixture was stirred thoroughly. The volume was then brought up to 200 μl per well with epithelial organoid culture medium containing 5% FBS. All operations were performed on ice. Next, 200 μl of the well-mixed cell-matrix gel suspension was added to each well of a 96-well low-adhesion cell culture plate pre-cooled to 4°C for 1 hour. This step was also performed on ice. The cell culture plate was then placed in a 4°C freezer for another 1 hour before being transferred to a 37°C cell culture incubator. The obtained endometrial organoids were cultured at 37°C for 7 days, with half the medium replaced every 2 days. Finally, endometrial organoids were induced with a specific drug for 3 days before harvesting.

[0042] The constructed human endometrial organoids were fixed, dehydrated, embedded, and ice-cut to a thickness of 5 μm. Immunofluorescence staining was performed, and the expression levels of αSMA and COL1A1 were used to demonstrate the anti-fibrotic effect of hesperidin. The results of hesperidin inhibiting fibrosis and promoting regeneration in complex human endometrial organoids are as follows: Figure 7 As shown in the figure, a represents the morphology of the constructed organoids, b represents the immunofluorescence staining of the organoids, c represents the fluorescence detection and statistical results of DAPI / α-SMA for hesperidin inhibiting fibrosis, d represents the fluorescence detection and statistical results of DAPI / COL1A1 for hesperidin inhibiting fibrosis, and e represents the fluorescence detection and statistical results of DAPI / KI67 for hesperidin promoting cell proliferation.

[0043] The results showed that human endometrial organoids were successfully constructed. Within the organoids, epithelial cells formed glandular structures surrounded by stromal cells. Similar to 2D culture, in complex human endometrial organoids, TGFβ1 stimulation significantly upregulated collagen deposition and α-SMA expression, while hesperidin attenuated the fibrotic-promoting effect of TGFβ1. Furthermore, hesperidin promoted proliferation-related KI67 expression at the organoid level, indicating that hesperidin possesses the ability to inhibit fibrosis and promote proliferation at the level of complex human endometrial organoids.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of hesperetin in the preparation of a drug for treating intrauterine adhesion.

2. Use of hesperetin in the preparation of a drug for preventing intrauterine adhesion.

3. Use of hesperetin in the preparation of a drug for promoting endometrial regeneration.

4. Use of hesperetin in the preparation of a drug for inhibiting endometrial fibrosis.

5. Use of hesperetin in the preparation of a drug for promoting endometrial tissue angiogenesis.

6. The use according to any one of claims 1 to 5, characterized in that, The hesperetin inhibits collagen deposition and expression of α-SMA.

7. The use according to any one of claims 1 to 5, characterized in that, The hesperetin up-regulates Hoxa10 and progesterone receptor, and improves endometrial receptivity.

8. The use according to any one of claims 1 to 5, characterized in that, The hesperetin promotes expression of CD31 and KI67.

9. The use according to any one of claims 1 to 5, characterized in that, The effective concentration of the hesperetin is 3-8 μM.

10. The use according to any one of claims 1 to 5, characterized in that, The drug further comprises pharmaceutically acceptable adjuvants.

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