Drug-loaded gel and application thereof in preparation of drug for repairing endometrial injury

Abstract

The invention discloses a drug-loaded gel and an application thereof in preparation of a drug for repairing endometrial injury, and belongs to the field of biological medicine, the drug-loaded gel is composed of poloxamer P407 and S100A8, the drug-loaded gel is prepared from PBS, and the ratio of the poloxamer 407 to the S100A8 to the PBS is 0.2 g: 5 [mu] g: 1 mL. According to the invention, poloxamer 407 hydrogel is used for carrying S100A8 to prepare a drug-loaded gel, and by constructing a rat endometrial injury model and applying the drug-loaded gel for treatment, the result shows that the drug-loaded gel can maintain the intact degree of endometrial epithelium and increase the levels of molecular indexes such as Ki-67, Claudin-1 and ZO-1 related to endometrial epithelium repair, so that the drug-loaded gel can be used for treating endometrial epithelium. Therefore, the endometrial injury of the rat is remarkably relieved, and a new idea is provided for repairing the endometrial injury.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a drug-loaded gel and application thereof in preparing a drug for repairing endometrial damage. Background Art

[0002] The uterus is the place where menstruation occurs and the fetus is conceived. The normal endometrium can repair and regenerate itself, and plays a vital role in female physiological and reproductive functions. However, intrauterine infection and intrauterine surgery can cause endometrial damage. If the damage cannot be repaired normally, the epithelium cannot re-cover the damage and the damaged endometrium may undergo fibrosis, scarring, and adhesion. At present, the treatment for preventing intrauterine adhesions mainly focuses on chemical drugs, physical barriers, stem cells, etc., but the effect of conventional treatments is still limited, and emerging treatments lack verification or have risks. Preventing intrauterine adhesions is still a difficult point. Therefore, continuously exploring the endometrial repair mechanism and finding effective factors for damage repair may provide a reference for improving the efficiency of endometrial damage repair.

[0003] S100A8 / A9 dimers or monomers can activate the repair function of stem cells, and promote the repair of skin wounds, myocardial injuries, dental pulp injuries, etc. by promoting angiogenesis, epithelial reconstruction, anti-inflammation, anti-fibrosis and other mechanisms, and have pleiotropic effects of injury repair. However, whether it can be beneficial to the repair of endometrial injuries has not been reported in the prior art. Summary of the invention

[0004] The purpose of the present invention is to provide a drug-loaded gel and its application in the preparation of a drug for repairing endometrial damage, so as to solve the problems existing in the above-mentioned prior art. Experimental verification shows that the drug-loaded gel prepared by the present invention can significantly alleviate endometrial damage in rats, providing a new idea for the repair of endometrial damage.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a drug-loaded gel, which consists of poloxamer 407 and S100A8.

[0007] Furthermore, the drug-loaded gel also contains PBS.

[0008] Furthermore, the ratio of the poloxamer 407, the S100A8 and the PBS is 0.2 g: 5 μg: 1 mL.

[0009] The present invention also provides a method for preparing the drug-loaded gel, comprising the following steps:

[0010] Poloxamer 407 was placed in PBS, stirred until it formed lumps, and then placed at 4°C for one night, and filtered to sterilize to obtain a poloxamer 407 solution;

[0011] The sterile S100A8 recombinant protein was dissolved in poloxamer 407 solution to obtain the product.

[0012] The present invention also provides application of the drug-loaded gel in preparing a drug for repairing endometrial damage.

[0013] The present invention also provides a medicine for repairing endometrial damage, which comprises the drug-loaded gel.

[0014] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0015] The present invention discloses the following technical effects:

[0016] The present invention uses poloxamer 407 hydrogel to carry S100A8 to prepare a drug-loaded gel, and the present invention constructs a rat endometrial injury model and administers the drug-loaded gel for treatment. The results show that the drug-loaded gel can maintain the integrity of the endometrial epithelium, increase the levels of molecular indicators related to endometrial repair such as Ki-67, Claudin-1, and ZO-1, thereby significantly alleviating rat endometrial damage, and providing a new idea for endometrial damage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Effects of S100A8 drug-loaded gel on the morphology of rat endometrium; A: HE staining of each group; B: endometrial thickness; C: number of glands; D: integrity of uterine cavity wall; E: number of endometrial crypts;

[0019] Figure 2 Effects of S100A8-loaded gel on proliferating cells in rat endometrium; A: Distribution of Ki-67-positive cells in endometrium; B: Number of Ki-67-positive cells in the entire endometrium; C: Number of Ki-67-positive cells in the uterine wall; D: Effect of S100A8 on the density of endometrial cells cultured in vitro;

[0020] Figure 3Effects of S100A8 on tight junctions of endometrial epithelial cells; A: Immunohistochemistry showed the distribution of Claudin-1 in the endometrium of each group, and the black arrows showed the aggregation of Claudin-1 under the cell membrane in the lateral contact area of ​​adjacent cells; the scale bar is 100μm; B: The expression of Claudin-1 in the endometrium; C: WB detection of the expression of ZO-1 in each group of endometrial cells; D: Immunofluorescence detection showed that Claudin-1 (red) and ZO-1 (green) were co-expressed in the endometrial cells of each group, and the cell nucleus was blue, and the scale bar was 200μm. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Example 1

[0027] 1. Construction of rat endometrial injury model

[0028] 1) Injury model: Six SD female unmated adult rats were used with synchronized estrous cycles and the model was established during the estrus period. The left uterus of the rat was selected, and a 0.3 cm longitudinal incision was made from the proximal end of the uterus. An endometrial scraper was inserted and the endometrium was scraped along the four walls of the uterus. After the endometrium was damaged, the incision was sutured and the abdomen was closed. The right uterus was used as a self-control.

[0029] 2) After 7 days, HE staining was performed to compare the endometrial integrity, endometrial gland number, fibrosis degree, and microvascular density of the injury model and the control group to evaluate the injury model. The results showed that the rat endometrial injury model was successfully established.

[0030] 2. Repair effect of hydrogel-loaded S100A8 on endometrial damage in rats

[0031] 1) Preparation of Poloxamer 407 Hydrogel Loaded with S100A8

[0032] 0.2 g of poloxamer 407 was placed in 1 mL of PBS, and the poloxamer 407 was stirred until it became a mass, and then placed in a 4°C refrigerator for one night, and sterile filtered to obtain a poloxamer 407 solution.

[0033] 5 μg of S100A8 recombinant protein was dissolved in poloxamer 407 solution to obtain a poloxamer 407+S100A8 mixed solution with a concentration of 5 μg / mL (ie, drug-loaded gel).

[0034] 2) Grouped medication

[0035] SD rats were randomly divided into 4 groups: blank control group (control), injured group (injured), gel group (Poloxamer407), and drug group (S100A8+P407), with 10 rats in each group. Each rat was injected with estrogen to synchronize the estrus cycle one day before surgery. After anesthesia was completed by intraperitoneal injection of 2% sodium pentobarbital (3mL / kg), the rat's abdominal cavity was opened. After the model was completed, 0.1mL of poloxamer 407+S100A8 mixed solution was injected into the uterus through the incision using a syringe. After standing for 1-2 minutes, the solution was turned from liquid to solid at body temperature, and then the syringe was withdrawn and the incision was sutured; the gel group was injected with only 0.1mL of poloxamer 407 solution according to the above method; the model group was injected with an equal amount of PBS; the other healthy uterine horn was used as a blank control group.

[0036] 3) Detection of damage repair effect

[0037] The uterus of rats in each group was taken out 7 days after treatment and observed by HE staining. HE staining showed the integrity of endometrial epithelium, the number of endometrial glands and the integrity of glandular epithelium.

[0038] At the same time, immunohistochemistry was used to detect endometrial repair-related molecular indicators such as cell proliferation (Ki-67) and cell junction (Claudin-1 / ZO-1) in each group.

[0039] 3. Effects of S100A8 on endometrial cells cultured in vitro

[0040] 1) Aseptically obtain 3-5 rat uteri, dissect longitudinally, and digest with trypsin at 37°C for 20 min. After digestion, scrape the uterine wall with a cell scraper. After collecting the cells, culture them for 5-7 days in primary culture, and the 2nd-4th generation cells are used for subsequent studies.

[0041] 2) The cells were treated with S100A8 and its inhibitors and divided into three groups: control group, drug-treated group (S100A8), and drug-treated + inhibitor group (S100A8 + FBS-ZM1). The cell density of each group was detected under a microscope, and the expression of tight junction proteins Claudin-1 and ZO-1 was detected by immunofluorescence.

[0042] Results Figure 1-3 ,Depend on Figure 1 As shown in Figure A, the endometrium in the injury group still had a large area of ​​damage that had not been repaired, the epithelial tissue was incomplete, and there was fluid accumulation in the uterine cavity; the endometrium in the poloxamer P407 group had columnar adhesion structures; the endometrial epithelial tissue in the poloxamer S100A8 group was repaired in an orderly manner, and the uterine cavity morphology was good. Figure 1 BE showed that the measurement results of different morphological indicators showed that there was no significant difference in endometrial thickness among the groups. The number of endometrial glands in the poloxamer P407 group and the poloxamer S100A8 group was not significantly different from that in the injury group. The integrity of the uterine cavity wall in the poloxamer P407 group and the poloxamer S100A8 group was significantly higher than that in the injury group, and there was no significant difference between the two groups. However, some areas of the uterine cavity wall of individual rats in the poloxamer P407 group were exposed by adhesion closure injury rather than relying on epithelial regeneration to improve integrity. The number of endometrial crypts in the poloxamer P407 group and the poloxamer S100A8 group was significantly less than that in the non-injured control group, and the number of endometrial crypts in the two groups was significantly higher than that in the injury group, and the poloxamer S100A8 group was significantly higher than the poloxamer 407 group, indicating that S100A8 in the uterine cavity significantly promoted the increase of damaged endometrial crypts.

[0043] Depend on Figure 2As shown in Figures AC, the number of Ki-67-positive cells in the endometrium of each injury group (injury group, poloxamer 407 group, and poloxamer carrying S100A8 group) was significantly higher than that of the control group, indicating that mechanical injury activated the regeneration of endometrial cells; the number of Ki-67-positive cells in the injury group was significantly higher than that in other groups. Comparing the number of Ki-67-positive cells per unit length of the uterine cavity wall (including intact epithelium and epithelial loss part) in each group, the number of Ki-67-positive cells in each injury group was still significantly higher than that in the control group; however, the number of Ki-67-positive cells in the cavity wall of the poloxamer carrying S100A8 group was higher than that of the poloxamer P407 group and the injury group. Primary endometrial cells were treated with S100A8 and its receptor inhibitor (FPS-ZM1) for extracellular effect, and DAPI fluorescent staining cells showed cell density. There was no significant difference in cell density among the control group, S100A8 group, and S100A8+FPS-ZM1 group. S100A8 did not significantly promote the proliferation of endometrial cells. Figure 2 (D) Although the number of Ki-67 positive cells in the injury group was the highest within the endometrium, the number of Ki-67 positive cells in the poloxamer-S100A8 group on the uterine cavity wall was the highest among all the samples. However, S100A8 did not significantly promote the proliferation of endometrial cells, suggesting that S100A8 promoted the migration and transformation of proliferating cells in the stroma to the epithelium.

[0044] Depend on Figure 3As shown in Figures AB, the intercellular tight junction protein Claudin-1 is highly concentrated in the lateral cell membrane contact area between epithelial cells under normal conditions (control group); however, in the case of endometrial damage (damage group), the integrity of epithelial tissue is difficult to maintain, and the intracellular distribution of the tight junction protein Claudin-1 between epithelial cells is chaotic; the tight junction protein Claudin-1 in the poloxamer P407 group and the poloxamer S100A8 group began to re-aggregate in the lateral contact area between epithelial cells; especially in the poloxamer S100A8 group, Claudin-1 was more orderly distributed. Under normal conditions, Claudin-1 is expressed in epithelial tissue, but after damage, cells with low expression of Claudin-1 also appear in the stroma near the damaged epithelium; the expression intensity of Claudin-1 in the endometrium (including epithelium and stroma) of each damage group was significantly higher than that of the control group, and epithelial damage promoted the expression of Claudin-1; the expression intensity of Claudin-1 in the poloxamer S100A8 group was significantly higher than that in other damage groups. Primary endometrial cells were cultured in vitro, and S100A8 and its receptor inhibitor (FPS-ZM1) acted on the cells. Immunofluorescence and WB were used to detect the effect of S1000A8 on tight junction proteins in endometrial cells. The results showed that the expression and distribution of two tight junction proteins, Claudin-1 and ZO-1, were synchronized in the cells; the expression of Claudin-1 and ZO-1 in the S100A8-treated group was significantly higher than that in the control group; the expression of Claudin-1 and ZO-1 in the FPS-ZM1-treated group continued to increase compared with the S100A8 group ( Figure 3 The above results indicate that S100A8 promotes the expression and orderly rearrangement of tight junction proteins, accelerating the formation of tight junctions between newborn epithelial cells.

[0045] Based on the above results, the drug-loaded gel prepared by the present invention can maintain the integrity of the endometrial epithelium, increase the levels of molecular indicators related to endometrial repair such as Ki-67, Claudin-1, and ZO-1, thereby significantly alleviating endometrial damage in rats.

[0046] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A drug-loaded gel, characterized in that: The drug-loaded gel consists of poloxamer 407 and S100A8.

2. The drug-loaded gel according to claim 1, characterized in that: The drug-loaded gel further comprises PBS.

3. The drug-loaded gel according to claim 2, characterized in that: The ratio of the poloxamer 407, the S100A8 and the PBS is 0.2 g: 5 μg: 1 mL.

4. The method for preparing the drug-loaded gel according to any one of claims 1 to 3, characterized in that: The following steps are involved: Poloxamer 407 was placed in PBS, stirred until it formed lumps, and then placed at 4°C for one night, and filtered to sterilize to obtain a poloxamer 407 solution; Take S100A8 and dissolve it in poloxamer 407 solution to obtain.

5. Use of the drug-loaded gel according to any one of claims 1 to 3 in the preparation of a drug for repairing endometrial damage.

6. A drug for repairing endometrial damage, characterized in that: The drug comprises the drug-loaded gel according to any one of claims 1 to 3.

7. The drug according to claim 6, characterized in that The drug further comprises pharmaceutically acceptable excipients.

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