CN111298104A

CN111298104A - Application of exenatide injection in medicine for treating and preventing intrauterine adhesion

Info

Publication number: CN111298104A
Application number: CN202010195092.9A
Authority: CN
Inventors: 王一青
Original assignee: First Hospital of Lanzhou University
Current assignee: First Hospital of Lanzhou University
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2020-06-19

Abstract

The invention relates to an application of exenatide injection in a medicine for treating and preventing intrauterine adhesion, wherein the dose of the exenatide injection is 10 ug/kg/d-200 mg/kg/d, the administration time is 7-28 d, the exenatide injection can improve the intrauterine adhesion degree of an IUA mouse, reduce collagen fiber deposition and promote the recovery of a gland, and in addition, the expression level of adhesion-related and inflammation-related genes (TGF- β 1, α -SMA and MMP-9) is reduced, so that the expression of adhesion factors and inflammation factors in the IUA mouse is inhibited, the intrauterine adhesion degree of the IUA mouse is obviously improved, the functions of intimal repair and gland regeneration are promoted, and the exenatide injection is expected to be used for treating and preventing intrauterine adhesion.

Description

Application of exenatide injection in medicine for treating and preventing intrauterine adhesion

Technical Field

The invention relates to the technical field of biology, in particular to application of exenatide injection in medicines for treating and preventing intrauterine adhesion.

Background

Intrauterine adhesions are also known AS Asherman Syndrome (AS). Fritisch was first described in 1894. In 1950, Asherman summarized 29 cases of intrauterine adhesion, and further detailed reports were made on the frequency, etiology and symptoms of intrauterine adhesion. Intrauterine adhesions often occur after trauma, infection, especially after birth injuries where estradiol levels are low. It is essentially the disease of endometrial fibrosis caused by damage to the basal layer of endometrium. The main complications of intrauterine adhesions are infertility, irregular menstruation (hypomenorrhea or amenorrhea), periodic pelvic pain, failure of pregnancy, and furthermore intrauterine adhesions may prevent blastocyst implantation, impairing the blood supply to the uterus and the early foetus, leading to infertility or recurrent abortion in patients with Asherman syndrome. According to a report of 2018, the prevalence rate of intrauterine adhesion is 4.6%, wherein hypomenorrhea accounts for 46.3%, and secondary infertility accounts for 70.7%; the artificial abortion history, uterine curettage history and postpartum hemorrhage history account for 56.1%, 51.2% and 31.7% respectively; according to classification, the prevalence of mild intrauterine adhesion was 34.1%, moderate 41.5%, and severe 24.4%.

TGF- β (transforming growth factor- β) is one of important fibrosis promoting factors, plays an important role in organ tissue fibrosis such as liver fibrosis, lung fibrosis, heart fibrosis and the like, and a plurality of experiments prove that TGF- β is also involved in the formation of the uterine cavity adhesion by regulating the expression of downstream genes.

In addition, the research shows that in the pulmonary fibrosis animal model and the human idiopathic pulmonary fibrosis, the local fibrosis lung tissue is not only TGF- β₁The level is increased, and myofibroblasts which express a large amount of smooth muscle actin α (α -SMA) are gathered, inflammatory cells secrete high-level TGF- β₁，TGF-β₁On one hand, the proliferation of the fibroblasts is inhibited, so that the fibroblasts stay at a certain phase of the intercellular phase, the synthesis of α -SMA mRNA and protein is accelerated, and the fibroblasts are further differentiated into the myofibroblasts, and on the other hand, the apoptosis of the myofibroblasts is inhibited.

Fibroblasts produce collagen, metalloproteinases (MMPs) and tissue inhibitors Thereof (TIMPs), wherein a deficiency in the pro-inflammatory factor MMP-9 (matrix metalloproteinase 9) improves wound healing, skin wounds are characterized by an excessive increase in MMP-9 and a decrease in TIMP 1 activity, i.e., an abnormally elevated MMP-9/TIMP-1 ratio. It was found that the MMP-9/TIMP-1 ratio was significantly reduced in fibroblast/myofibroblast colony derived medium from rats pretreated in vitro with exendin-4 (P < 0.001).

Exenatide (exenatide) is an artificial synthetic product of glucagon-like peptide-1 (GLP-1) analogue exendin-4 and consists of 39 amino acids. The preparation is mainly used for improving the blood sugar control of type 2 diabetes patients, and is suitable for patients who only use metformin, sulfonylureas and metformin and sulfonylureas, and have poor blood sugar control. Meanwhile, in addition to the effect of exenatide in metabolic diseases, a large number of experiments prove that the exenatide has good curative effect in the aspect of anti-fibrosis, but the research of the exenatide in the aspect of treating intrauterine adhesion is not found at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of exenatide injection in the medicines for treating and preventing the intrauterine adhesion.

In order to solve the problems, the application of the exenatide injection in the medicines for treating and preventing the intrauterine adhesion is characterized in that: the exenatide injection is used in an amount of 10 ug/kg/d to 200 mg/kg/d, and the administration time is 7 d to 28 d.

The dosage of the exenatide injection is 50 ug/kg/d, and the administration time is 14 d.

Compared with the prior art, the invention has the following advantages:

the invention establishes a uterine cavity adhesion mouse model by the stimulation of dual functions of mechanical injury and Lipopolysaccharide (LPS) inflammation. And administered in groups without intervention with doses of exenatide. The research finds that: exenatide can improve the intrauterine adhesion degree of IUA mice and reduce the deposition of collagen fibers (PLess than 0.05) and can promote the recovery of glands, in addition, the expression level of adhesion-related and inflammation-related genes (TGF- β 1, α -SMA and MMP-9) is reduced, so that the expression of adhesion factors and inflammation factors in IUA mice is inhibited, the intrauterine adhesion degree of the IUA mice is obviously improved, the functions of intima repair and gland regeneration are promoted, and the intrauterine adhesion can be treated and prevented.

The following experimental methods are all conventional methods unless otherwise specified.

⑴ purchase and rearing of laboratory animals:

the animal experiment strictly complies with the animal experiment center operating specifications of Lanzhou university. 54C 57BL/6 female mice with the size of 8W and the weight of 16-18 g/mouse are purchased from Beijing Wintolite laboratory animal center, and are bred in animal laboratories of Lanzhou university college of medicine, the temperature is controlled within 22 +/-2 ℃, the humidity is 70%, the breeding is carried out under the environment with the photoperiod of 12h, and the mice are fed with free diet and drinking water in the whole experimental process.

⑵ reagents and instruments required for the experiment, as shown in tables 1 and 2:

TABLE 1 reagents required for the experiment

TABLE 2 instruments required for the experiment

Preparation of related solution:

i preparation of 6mg/L lipopolysaccharide solution:

0.6mg of lipopolysaccharide is weighed and dissolved in 10mL of physiological saline to prepare a lipopolysaccharide solution with the concentration of 60mg/L, 2mL of the lipopolysaccharide solution with the concentration of 60mg/L is taken out to be diluted by 10 times, and then the lipopolysaccharide solution with the concentration of 6mg/L required by the experiment is prepared.

Ii, preparing an exenatide solution:

6 mice in the high dose group of 50ug/kg exenatide were dosed according to 30g, the dose was 20ug/kg, and the total daily dose was: 0.03kg × 50ug/kg × 6=9 mg;

6 mice in 10ug/kg middle dose group of exenatide were dosed according to 30g, the dose is 10ug/kg, and the total daily dose is: 0.03kg × 10ug/kg × 6= 1.8 mg;

6 mice in the low dose group of 2ug/kg exenatide were administered at a dose of 5ug/kg per 30g, and the total daily dose was: 0.03kg × 2ug/kg × 6=0.36 mg;

therefore, the total daily dose of the high, medium and low dose 3 groups of mice was:

9mg+1.8mg+0.36mg=11.16mg。

research proves that 7 days after the rabbit IUA model is built, the surface of the endometrium is completely arranged by epithelial cells, and the regenerative cells are flattened (60% -70%), cuboidal or low-columnar. Few glands were visible and stroma was highlightedMore fibrous tissue, collagen aggregation, focal bleeding, hyperemia, leukocyte infiltration, some newly formed capillaries were observed within 14 days, the uterine matrix showed a small amount of polymorphonuclear leukocytes and macrophages containing ferrihemoflavin, showing interstitial fibrosis and collagen aggregation, and after 28 days the histological reconstruction of the endometrium was essentially complete, with fibrosis being observed in a small number of interstitial regions [ YANG W, LAI X, WEIMIN D, et al]Plos One, 2015, 10(5): e 0126249. Thus, the administration cycle for the mice in this experiment was set to 14 days, 1 time daily for a total of 14 administrations, so that the total dose throughout the administration was: 11.16mg × 14=156.24mg, and 160mg of the drug was weighed out to avoid loss during drug formulation. Dissolving 160mg of the drug in dimethyl sulfoxide (DMSO) (in principle, the amount of DMSO is not more than 1% of the total volume of the drug), dissolving the drug in DMSO completely, and sterilizing ddH₂And O, diluting the dissolved medicines into three administration concentrations according to the requirements of experiments, finally subpackaging the medicines with the concentrations into sterilized EP tubes, sealing, marking, and storing at-20 ℃ for later use.

⑶ animal grouping and dosing regimen:

36C 57BL/6 female mice 19 weeks old were grouped as shown in Table 3:

TABLE 3

The low, medium and high dose groups are injected subcutaneously once a day from the first day after the intrauterine adhesion; the model group was injected subcutaneously with ddH from the first day after intrauterine adhesion₂O is taken once a day; the normal group and the sham-operated group were normally kept without any special treatment. The change of the vital signs of the mice is observed every day in the administration process, the weight is weighed and recorded, the administration period is 2 weeks, and the materials are obtained later for subsequent experiments.

⑷ mouse model making:

selecting 36 mice qualified for quarantine, randomly dividing the mice into 6 groups, balancing the body weight by about 17-19 g after One week of environmental balance, performing an operation in an animal experiment center after the preoperative preparation of 20 gonadotropins in pregnant horse serum is completed before 36h of each mouse in the same estrus period, simulating a mechanical and infection combined modeling method (reported in the literature as [ YANG W, LAI X, WEIMIN D, et al. Exenation Reses Endothelium-Dependentraxation in Rat) after the preoperative preparation of 20 IU. is completed, simulating a mechanical and infection combined modeling method (A Novel organ implanted by High Glucose: A Novel organ intensive PPAR β -NO Signaling Pathway J. 2015. 10(5 e 0126249) and a preoperative abdominal wall of a mouse after the intraperitoneal injection of a pentosan needle is completed, after the intraperitoneal injection of a medicament into the abdominal cavity, after the abdominal cavity is filled into the abdominal cavity, injecting a pentosan needle catheter, injecting a serous catheter needle into the abdominal cavity, injecting a serous uterine needle, injecting a serous needle, injecting a serous needle, a needle.

⑸ paraffin sections of mouse uterine tissue, hematoxylin-eosin staining (H & E), Masson staining, immunohistochemical staining:

preparation of paraffin section of mouse uterus tissue includes fixing ① uterus tissue, removing fat and other tissues around uterus, taking 5mm uterus tissue, putting it in 4% paraformaldehyde solution, standing overnight at room temperature, dewatering ② uterus tissue, putting it in 50% alcohol for 1.5 hr, 70% alcohol for 1.5 hr, 85% alcohol for 1.5 hr, 95% alcohol for 1.5 hr, 100% alcohol for 1 hr, making it transparent ③ uterus tissue, mixing alcohol solution and xylene solution, putting uterus tissue in the mixture for 1 hr, putting it in xylene solution for 1 hr, putting it in new xylene solution for 1 hr, penetrating ④, putting the xylene solution and paraffin solution in the same volume, putting the uterus tissue in the mixture for 1.5 hr, putting it in 62 deg.C for 2 hr, putting it in new paraffin for 2 hr, filling in mould, fixing paraffin solution, baking, cutting it in 82 μm paraffin section, baking, cutting, slicing, baking, and baking.

H & E staining of mouse uterine tissue:

h & E staining solutions were prepared as in table 4:

TABLE 4H & E staining solution formula

① white slices are inserted into a staining rack one by one and are placed in a 60 ℃ oven for 1h, ② dewaxing is carried out, the staining rack is placed into a staining jar containing different experimental reagents according to the dewaxing step for dewaxing step by step and is placed into xylene I, the duration is 20min → xylene II (the concentration and the volume of the xylene I are the same), the duration is 20min → absolute ethanol I, the duration is 15min → absolute ethanol II (the concentration and the volume of the absolute ethanol I are the same), the duration is 15min → 95% ethanol, the duration is 10min → 90% ethanol, the duration is 5min → 80% ethanol, the duration is 5min → placing into tap water for slow washing, the duration is 5min, ③ staining is carried out, the staining rack is placed into a staining jar added with a sappan wood reagent for a duration, the staining rack is soaked for a duration of 7-8, the staining jar is placed into a staining jar containing tap water for a few seconds, the staining rack is placed into a hydrochloric acid ethanol solution for slow washing, the staining rack is placed into a staining jar, the rack is placed into tap water → the rack for slow washing, the staining, the rack is placed into a rack for slow washing, the dyeing rack is placed into a rack for slow washing, the rack is placed into a rack with a rack, the rack is placed into a rack for slow washing rack, the dyeing rack is placed for slow washing rack with a slow washing time length of drying time, the dyeing rack with a rack;

iii Masson staining of mouse uterine tissue:

masson staining solutions were prepared as in table 5:

TABLE 5 Masson staining solution formula

The staining method comprises the steps of ① placing paraffin sections one by one on a staining rack, placing the paraffin sections in an oven for 60 min, placing the paraffin sections in a staining cylinder filled with a Bouin solution for soaking (at room temperature overnight), washing the paraffin sections in a staining cylinder filled with a celestine solution for the second day by running water (so as to avoid excessive washing of tissues on a glass slide) until no yellow staining solution exists on the sections, ② placing the staining rack in the staining cylinder filled with celestine for soaking for 2-3 min by using water for washing slightly, ③ placing the paraffin sections in the staining cylinder filled with a Mayer haematoxylin a staining cylinder filled with a Mayer haematoxylin solution for dyeing for 2-3 min, placing the staining rack in the staining cylinder filled with an acidic ethanol differentiation solution for differentiation for 2s, placing the staining rack in the staining cylinder filled with a running water for differentiation for 2s, then placing the staining cylinder filled with a running water for slow washing by running water for washing the tissues on the glass slide for washing for soaking for 2min (so as to avoid excessive washing of tissues on the glass slide), placing the staining rack in the staining cylinder filled with a distilled water for slow washing for soaking for 2s, after the staining solution for 2s for drying for 2min, after soaking the staining for ⑥ min, placing the neutral staining solution for soaking of the neutral staining rack for 355 min, after the neutral staining solution for soaking of the neutral staining solution for a staining for 10min, after the neutral staining solution for a staining solution, after the neutral staining solution for a period of a staining solution for a period of a staining procedure, after the neutral staining procedure of a staining procedure.

Iv immunohistochemical staining of uterine tissue:

① baking and dewaxing, placing paraffin slice on staining rack, baking and dewaxing together with H&E staining, ② removing wax, taking out the staining rack with the section, and placing in ddH₂Soaking in O beaker for 5min, ③ repairing, fixing the slices on slicing rack, and adding citric acid buffer solution (0.4 g citric acid +3.0g sodium citrate + ddH)₂Fixing O volume to 1L, adjusting pH to =6.0 with 4mg/L NaOH), adding citric acid buffer solution to 2/3, wrapping the beaker with tinfoil in dark place, heating at high temperature and high pressure for 15min (to save time), immediately lowering pressure, taking out the beaker, and cooling to room temperature (to remove tinfoil), adding 3% H to ④₂O₂Soaking at room temperature for 10min (to block endogenous peroxidase activity), washing with 1 XPBS (on a shaker) for 3 times, each for 5min, blocking ⑤ by placing the slices one by one in a wet box and incubating with ice 1% BSA (30 uL drop for each tissue) at room temperature for 1h, after incubation, wiping off excess fluid around the tissue one by one with a paper towel, incubating primary antibodies ⑥ by diluting the primary antibodies with 1% BSA ice (TGF- β 1:1% BSA =1:500; α -SMA:1% BSA =1:200; MMP-9:1% BSA =1: 500), placing primary antibodies 30uL drop for each tissue overnight at 4 deg.C, rewarming ⑦ by removing the wet box from the refrigerator and leaving it at room temperature for 1h, incubating secondary antibodies ⑧ by 1 XP washing the slices 2 times, each for about 3min, gently swinging the slices and carefully wiping the fluid around the uterine tissue with a paper towelDripping diluted secondary antibody (secondary antibody goat anti-rabbit: 1% BSA =1: 200), dripping 30uL of secondary antibody into each tissue, incubating for 1h in a wet box at 37 ℃, fixing the section on a section frame, washing for 2 times by 1 XPBS (phosphate buffer solution) for 3min each time, ⑨ DAB developing, preparing DAB developing solution on ice and in a dark place (buffer solution: solution A: solution B =1000:50: 50), dripping 30uL of DAB developing solution into each tissue to completely cover the tissue, determining the tissue developing degree under a microscope, and dripping ddH (ddH) into the other tissue when the developing degree of one tissue is observed if the left and right primary tissues are different₂O protection), placing in hematoxylin staining jar for 25s (hematoxylin can stain ribose in cell nucleus and cell into bluish purple color), washing with tap water for 5min, differentiating with 1% hydrochloric acid for 2s, washing with tap water for 5min, dehydrating ⑩, and sealing&E, dyeing.

⑹ extraction of mouse uterine tissue total RNA and Real time PCR

① taking out the mouse uterine tissue from a refrigerator at-80 deg.C, placing it on ice, shearing 3mm long uterine tissue, washing it twice with PBS, sucking water from the surface of the tissue with filter paper, weighing with tinfoil, labeling, placing it in an enzyme-free grinding enzyme-free EP tube (all EP tubes and centrifuge tubes used in PCR experiment are treated with DEPC water), placing 1 big 2 small grinding steel balls into the tube, adding 1mL Trizol and precooling grinding base plate (-20 deg.C), placing the tissue in a Servicebio grinder, covering the cover, grinding at 70Hz for 90s, fully blowing with a pipette (without temporary storage in a refrigerator at-80 deg.C), adding 200 μ L chloroform into ②, shaking for 1min, standing at room temperature for 3min, centrifuging at 4 deg.C (12000 × 15 min), separating the sample into three layers, the upper layer is an organic layer, the middle layer is an aqueous layer, the colorless aqueous layer is collected in a colorless aqueous layer, and the colorless aqueous layer is adsorbed by a centrifuge tube, and the colorless aqueous layer is adsorbed by a colorless aqueous layer, and the colorless aqueous layer is adsorbed by a colorless liquid crystal gel-free layer, and the colorless aqueous layer is adsorbed by a slow centrifuge tube, and the colorless aqueous layer is adsorbed by the colorless organic layer, and the colorless aqueous layer, the colorless aqueous layer is adsorbed by the colorless liquid centrifuge tube, and the colorless tube, the colorless aqueous layer is adsorbed by the colorless tube, the colorless liquid centrifuge tube, and the colorless tube is adsorbed by the colorless tube, and the colorless tube, the colorless tubeMixing with isopropanol, standing at room temperature for 10min, centrifuging at 4 deg.C (12000 rpm × 10 min), observing white flocculent precipitate on tube wall or tube bottom, discarding supernatant, adding 1mL 75% ethanol (DEPC water: anhydrous alcohol =1: 3) into each tube, vortexing with a vortex machine, washing white precipitate, centrifuging at 4 deg.C (12000 rpm × 5 min), opening EP tube cover, discarding supernatant, evaporating residual liquid at room temperature, air drying, and adding 20 μ L RNase-freeH₂And completely dissolving the precipitate by using O, and using the completely dissolved precipitate for preparing cDNA subsequently.

① preparing cDNA by reverse transcription of RNA, measuring the concentration and absorbance of the extracted RNA with a Nanodrop 2000 spectrophotometer to ensure the purity of RNA, and ② measuring the RNA with RNase-free ddH₂Adjusting to the same concentration for subsequent use, ③ establishing 20 μ L reaction system using FastKing RT Kit (With gDNA) from TIANGEN corporation 50 ng-2 μ g total RNA, thawing template RNA on ice, thawing 5 XgDNAbuffer, FQ-RT Primer Mix, 10 XKing RT Buffer, RNase-Free ddH at room temperature₂The following experimental steps should be carried out on ice, in order to accurately prepare reaction liquid, each liquid should be prepared into Mix firstly, and then the Mix is filled into each reaction tube in the same volume, ④ the following steps and the proportioning scheme in the table 6 are used for preparing mixed liquid, aiming at removing genome DNA, fully mixing, centrifuging, then placing the mixed liquid in a gradient PCR instrument 42 ℃, incubating for 3min, and then placing the mixed liquid on ice:

TABLE 6 gDNA removal reaction System

⑤, placing on ice after mixing well, preparing reverse transcription system mixture 2(10 μ L) according to table 7:

TABLE 7 reverse transcription System

⑥ adding Mix in reverse transcription reaction into reaction solution of gDNA removal step, mixing well, incubating at 42 deg.C for 15min in gradient PCR instrument, incubating at 95 deg.C for 3min in ⑦ gradient PCR instrument, and placing on ice to transcribe cDNA for subsequent experiment, or storing at-40 deg.C for use.

Real Time PCR: establishing a Real-Time PCR reaction system: lysis of 2 XSuperReal PreMixPlus, 50 XROX Reference Dye, template, primer and RNase-free ddH₂O, balancing all reagents at room temperature and fully mixing the reagents; then, the Real-Time PCR reaction system was prepared on ice, and 2 XSuperreal PreMixPlus and 50 XROX Reference Dye were stored in the dark.

TABLE 820 μ l preparation of the reaction systems

After preparing the system shown in Table 8 in a dark room, the reaction tube is covered, the reaction tube is gently mixed and centrifuged, a centrifuge is used for centrifuging all the components to the tube bottom of the reaction tube for a short time, the reaction system is placed in an Aglient Mx3000P fluorescence quantitative PCR instrument according to the sample adding sequence for reaction, a three-step reaction method is adopted, wherein ① pre-denaturation is carried out at 95 ℃ for 15min, 1 cycle number (the temperature and the time must be set for fully activating the heat-start enzyme), ② PCR reaction is carried out at 95 ℃ for 10s (denaturation), 20 s at 55 ℃ for 20 s (annealing), 20 s at 72 ℃ for 20 s (extension), and the sequence of the used primers (Shanghai biological engineering) is shown in Table 9.

TABLE 9 primer sequences

⑺ statistical analysis:

all data in the experiment are statistically analyzed by using Excel2010, SPSS22.0, Graphpad prism6.0, Image-pro Plus6.0 and software, and the results are in the form of mean value plus or minus standard deviation. By usingtThe test is performed for an intra-group comparison,P<0.05 statistical differences were considered between the data；P<0.01 considered the statistical differences between the data to be significant.

⑻ results of the experiment:

① Effect of exenatide on body weight and intimal gland number of mice with intrauterine adhesion:

i gross specimen observation:

after the dosing (14 day) cycle was complete, the mice were sacrificed by cervical dislocation. The uterus surface of the mice in the normal group is ruddy, uniform in thickness, good in elasticity, smooth in surface and easy to separate without adhesion with surrounding tissues when dissected. Mice in the IUA model group have pale uterus surfaces, obvious edema, coarseness, poor elasticity, narrow uterine cavities, even occlusion and serious separation difficulty of uterus and surrounding tissue adhesion. In mice of the IUA model and the administration group, the surface of the uterus is slightly light, part of uterine cavity is edematous, the elasticity is poor, and the uterus is slightly adhered to surrounding tissues.

Ii Effect of Exenatide on weight gain in mice with intrauterine adhesions:

the weight gains were compared between groups (see table 10), from the first day of modeling, marked as day 0, to the end of exenatide treatment, and weights were weighed every other day and recorded until after the end of day 14 treatment. Control group: 1.443 ± 0.3156; the sham operation group: 1.290 ± 0.3968; model group: 1.300 ± 0.2301; exenatide low dose group (2 ug/kg): 1.135 ± 0.2420; exenatide middle dose group (10 ug/kg): 1.868 ± 0.2912; exenatide high dose group (50 ug/kg): 1.608 ± 0.1535; no statistical differences were observed between the 6 groups of data (P＞0.05）。

TABLE 10 Effect of Exenatide on body weight of groups of mice

Iii H & E staining for endometrial visualization:

h & E stained 40-fold and 200-fold, and endometrial morphology was observed under a microscope (see fig. 2). Normal group: the mouse endometrium, muscle and serosa are complete; the intima tissue can be seen into regularly arranged single-layer columnar epithelium, the interstitium has no edema, and the glands are uniformly distributed; the interstitial cells are arranged tightly into oval shape, and the nucleus is deeply dyed. IUA model group: the thickness of the endometrium of the mouse is thinned, the plica is reduced, and partial lumens are reduced or even disappear; the number of glands is obviously reduced, and the glands are unevenly distributed; occasionally, leukocyte infiltration occurs, and interstitial edema is obvious. Exenatide low dose group (2 ug/kg): the intima tissue is relatively complete, and the intima becomes thin; interstitial edema and maldistribution of glands. Exenatide middle dose group (10 ug/kg): the endometrium tissue is intact, the endometrium is thinner than the normal tissue, the interstitial edema is reduced, the lumen is normal, the fold and the depression can be seen, and the number of glands is increased. Exenatide high dose group (50 ug/kg): the endometrium is thinner than the normal group, the epithelial tissue is more complete and is in a single-layer columnar epithelium, the interstitial edema is lighter, the lumen is normal, the plica and the depression can be seen, and the number of glands is obviously increased.

Influence of iv Exenatide on the number of endometrial glands in mice with intrauterine adhesion:

observing the tissue sections under a microscope high power microscope, observing and counting endometrial glands according to the sequence of each section from left to right and from top to bottom, repeatedly counting 3 times for each section, and calculating the average value. As a result, it was found that: endometrial gland counts were compared between groups (see table 11). Control group: 14.67 +/-1.043; the sham operation group: 17.33 ± 0.7454; model group: 8.000 +/-1.193; exenatide low dose group (2 ug/kg): 15.00 +/-5.321; exenatide middle dose group (10 ug/kg): 13.89 ± 4.418; exenatide high dose group (50 ug/kg): 19.78 ± 2.278; the control group has no statistical difference compared with the sham operation group (14.67 plus or minus 1.043 vs17.33 plus or minus 0.7454); compared with the normal group (8.000 +/-1.193 vs 14.67 +/-1.043), the number of endometrial glands in the model group is obviously reduced, and the statistical significance is achieved (P is less than 0.01); the number of glands in the low dose of exenatide compared to the model group (15.00 + -5.321 vs 8.000 + -1.193) was increased, but not statistically significant; the number of glands in the dose group in exenatide is increased compared with that in the model group (13.89 +/-4.418 vs 8.000 +/-1.193), but the number of endometrial glands in the dose group is not statistically significant; the number of glands in the exenatide high dose group was significantly increased and statistically significant (P < 0.01) compared to the model group (19.78 ± 2.278 vs 8.000 ± 1.193).

TABLE 11 Effect of Exenatide on the number of endometrial glands in IUA mice

Note: model group to normal group comparison: p <0.01, IUA model + exenatide (50 ug/kg) compared to model group.

② Effect of Exenatide on fibrosis in mice with intrauterine adhesions:

i Masson staining:

masson staining mainly exhibited differences in collagen fibers and muscle fibers. By staining the collagen fibers in the fibrous tissue to be blue, the muscle fibers, keratin, cellulose, cytoplasm, etc. to be red, the nucleus to be blue brown. Therefore, the degree of fibrosis can be judged according to the stained area of the collagen fiber tissue.

Masson staining results showed: 3 different visual fields are randomly selected for each tissue under a high power microscope to be photographed, the collagen fiber coloring area is measured by adopting Image Pro Plus, and the measured data is statistically analyzed through Excel2010 and Graphpadprism 6.0. The results are shown in FIG. 3: compared with the false operation group, the difference of blue areas in endometrium is smaller in the normal group, and no statistical difference exists; the uterine wall of the model building group becomes thin, the endometrium is replaced and covered by fibrous scar tissues, the blue area in the endometrium is larger, the deposition of collagen fibers is increased, and is obviously more than that of a normal control group, and the significant difference exists (the)P< 0.001); each concentration gradient (2 ug/kg, 10ug/kg, 50 ug/kg) exenatide treatment group: the deposition of collagen fibers in the visible part of endometrium is obviously reduced compared with the model group, and the deposition of collagen fibers is gradually reduced along with the increase of the concentration of the drug, and the 50ug/kg exenatide treatment group has statistical significance (the step of treating the endometrium by the injection is shown in the specification and the step of treating the endometrium by the injection is shown in the specification (the step of treating the endometrium by the injection is shownP＜0.01）。

Ii effects on uterine tissue protein expression:

in order to further study the expression levels of TGF- β 1, α -SMA and MMP-9 proteins in the uterine tissue of IUA mice, each was collectedMouse uterine tissue of group, paraffin section of control group, sham operation group, model group and exenatide concentration gradient treatment group is immunohistochemical (figure 4A), analysis and comparison of TGF- β 1, α -SMA and MMP-9 protein content in uterine tissue are performed, each section is randomly selected to be 3 visual fields at 200 times, positive staining area is analyzed by Image-Pro Plus6.0 software, optical density value (IOD) is calculated, and finally statistical analysis is performedPTGF- β 1 is mainly located in cytoplasm and can be found to be positively expressed in endometrium gland epithelium and mesenchyme, which indicates that TGF- β 1 signaling is activated in the cells, the exenatide low-dose treatment group has reduced but no difference compared with the model group in the expression amount of TGF- β 1, and the medium-dose group has reduced and has difference compared with the model group in the TGF- β 1 (x)P< 0.05 fig. 4B), the high dose group had significant difference compared to the model group (. star)P<0.01 FIG. 4B). α -SMA expressed in mesenchymal cells and exhibited dark brown positive stained areas in cytoplasm, and the increase in expression of α -SMA in the IUA model group was significantly different from that in the normal group (. about.. aboutP<0.001 fig. 4C), there was a significant difference between the exenatide high dose treatment group and the model group (. + -.)P<0.01 FIG. 4C), but there was no significant difference between the model group and the exenatide low dose treatment group. MMP-9 as an inflammation medium, the expression amount of the MMP-9 is significantly different in the normal group compared with the model groupP<0.001 FIG. 4D), but the expression level did not decrease in the low and medium dose treatment groups of exenatide, while the expression level decreased in the high dose treatment group of exenatide and had no difference from the model group (.)P< 0.05 FIG. 4D); overall, the above data further confirm the key role of exenatide in the treatment of IUA mice.

Iii Effect on uterine tissue-associated Gene expression:

in order to research the expression of TGF- β 1, α -SMA and MMP-9 gene level in the uterine tissue of normal mice, IUA mice and exenatide treatment groups of mice, the mice of each group are collectedExtracting total mRNA from uterine tissue, detecting expression levels of genes related to fibrogenesis and inflammatory factors in uterine tissue of mouse with uterine cavity adhesion by qPT-PCR (as shown in figure 5), comparing expression levels of TGF- β 1, α -SMA and MMP-9 in normal group with that in model group, respectively, finding that the expression levels of TGF- β 1, α -SMA and MMP-9 in model group are increased, and there is a statistically significant difference (a. about.P<0.001 FIG. 5.) thereafter, when the low, medium and high dose treatment groups of exenatide were compared with the same model group, it was found that the expression level of the fibrogenic factor TGF- β 1 was decreased in the high dose treatment group of exenatide, and there was a statistical difference (. lambda.) (compared with the model group)P<0.01, FIG. 5A), the medium and high dose groups can inhibit the expression of α -SMA in the mice with intrauterine adhesion, so that the expression level of the gene in the mouse tissues is reduced, and the high dose group is compared with the model groupP<0.01, FIG. 5B) with significant statistical differences; the expression level of MMP-9 was reduced in the high exenatide treatment group compared to the model group (a)P< 0.05, fig. 5C), but there was no significant difference.

In conclusion, exenatide can inhibit the expression level of the fiber-related factor gene.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a diagram showing the steps of molding IUA mice of the present invention (A → F).

FIG. 2 shows the results of tissue transection of mice 14 days after IUA of the present invention, and H & E staining (40-fold and 200-fold).

FIG. 3 is a graph showing the effect of exenatide of the invention on endometrial fibrosis in various groups of mice. (A) Masson trichrome staining of collagen (40 x and 200 x) 14 days after IUA, histological structure of uterus in each group. (B) Results of analysis of endometrial fibrosis regions in each group 14 days after IUA.

FIG. 4 shows the effect of exenatide of the present invention on the expression level of tissue proteins in IUA mice, (A) immunohistochemistry of TGF- β 1, α -SMA, and MMP-9 after IUA mice were treated with different doses of exenatide for 14 days, (B) quantitative analysis of immunohistochemistry of TGF- β 1, (C) quantitative analysis of immunohistochemistry of α -SMA, (D) quantitative analysis of immunohistochemistry of MMP-9.

FIG. 5 is a graph showing the effect of exenatide of the present invention on RNA expression in IUA mouse tissue, (A) the result of quantitative analysis of TGF- β 1 expression, (B) the result of quantitative analysis of α -SMA expression, (C) the result of quantitative analysis of MMP-9 expression.

In the figure, P is less than 0.05, P is less than 0.01, P is less than 0.001, IUA is intrauterine adhesion, TGF- β 1 is transforming growth factor- β 1, α -SMA is α -smooth muscle actin, and exenatide is exenatide.

Detailed Description

Example 1 application of exenatide injection in the preparation of medicines for treating and preventing intrauterine adhesion: the exenatide injection has the dosage of 10 ug/kg/d and the administration time of 28 days.

Example 2 application of exenatide injection in the preparation of medicines for treating and preventing intrauterine adhesion: the exenatide injection has the dosage of 50 ug/kg/d and the administration time of 14 d.

Example 3 application of exenatide injection in the preparation of medicines for treating and preventing intrauterine adhesion: the exenatide injection is used in an amount of 200 mg/kg/d for 7 d.

In the above examples 1 to 3, the exenatide injection is provided by exenatide acetate provided by gill biochemical (shanghai) ltd or Sigma.

Claims

1. The application of the exenatide injection in the medicines for treating and preventing the intrauterine adhesion is characterized in that: the exenatide injection is used in an amount of 10 ug/kg/d to 200 mg/kg/d, and the administration time is 7 d to 28 d.

2. The use of exenatide injection according to claim 1 in a medicament for the treatment and prevention of uterine cavity adhesions, characterized by: the dosage of the exenatide injection is 50 ug/kg/d, and the administration time is 14 d.