Enzyme response hydrogel for treating inflammatory bowel disease as well as preparation method and application of enzyme response hydrogel

Abstract

The invention discloses an enzyme response hydrogel for treating inflammatory bowel disease as well as a preparation method and application thereof, and the enzyme response hydrogel comprises the following components in percentage by weight: 13-18% of an effective part of pediatric stolonifera, 75-85% of an enzyme response gel matrix, 0.5-1% of purified water, 2-4% of a transdermal absorption enhancer, 0.5-1% of a solubilizer and 0-0.1% of a preservative. The enzyme response gel matrix is tripolyglycerol monostearate, the transdermal absorption enhancer is at least one of azone, oleic acid and propylene glycol, the solubilizer is at least one of tween-80, propylene glycol, polyethylene glycol and span, and the preservative is at least one of ethylparaben, phenoxyethanol and hexanediol. The enzyme response hydrogel prepared by the invention can release drugs in the presence of a specific enzyme, and due to the characteristic, the enzyme response hydrogel can act on diseased regions more accurately in treatment of inflammatory bowel diseases, so that the side effects of the whole body are reduced, and the safety and effectiveness of treatment are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an enzyme-responsive hydrogel for treating inflammatory bowel disease, and a preparation method and application thereof. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing, and difficult-to-treat autoimmune disease, primarily encompassing Crohn's disease and ulcerative colitis. While the etiology and pathogenesis of IBD remain incompletely understood, existing studies have shown that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a significant role in the development of IBD, including intestinal mucosal damage, submucosal matrix destruction, and even complications such as perforation, fistula formation, and cancer.

[0003] Matrix metalloproteinases (MMPs) belong to the M10 matrix protein subfamily of the zinc protease family. They are secreted by monocytes, macrophages, inflammatory T lymphocytes, etc. and can specifically degrade the extracellular matrix (ECM), promote cell apoptosis, affect angiogenesis, promote cytokine release, and participate in the occurrence and development of IBD and its complications through multiple pathways. Studies have found that the levels of some MMPs increase with the aggravation of IBD or the development of complications. Therefore, it is of great significance to design an intelligent hydrogel system that releases drugs according to fluctuations in IBD disease. The decomposition of triglycerol monostearate is positively correlated with the concentration of MMPs. When prepared into a hydrogel, it can be degraded according to the concentration of MMPs, thereby promoting drug release and achieving on-demand drug delivery.

[0004] Swertia patens Burk. is a dried whole herb of Gentianaceae Swertia patens, a plant of Swertia patens Burk., which is a traditional medicinal material used by the Yi ethnic group in Yunnan Province. It is used for the treatment of gastrointestinal diseases such as functional abdominal pain and enteritis, and is safe and effective. However, in the prior art, Swertia patens is generally used directly as a raw material to form a Chinese medicine composition with other Chinese medicine raw materials. It is directly decocted and taken during use, which not only increases the pain of the patient taking the medicine, but also requires decocting each time it is taken, and the operation is complicated. In addition, the treatment time is long, and the therapeutic effect is also not good. This is mainly because the decocting process cannot extract the effective ingredient of Swertia patens, and the effective ingredient obtained by decocting cannot effectively reach the focus by directly taking it. For example, patent CN104069419A discloses a traditional Chinese medicine composition for treating acute and chronic gastritis and duodenal ulcers, which includes Liushenqu, fennel, elecampane, galangal, licorice, galangal, herba scutellariae, tsaoko, cyperus rotundus, chrysanthemum indicum, tangerine peel, notoginseng, chicken gizzard lining, tangerine peel, houttuynia cordata, betel nut, patchouli, amomum villosum, fructus aurantii, chuanxiong and atractylodes macrocephala. The composition involves the use of herba scutellariae, but the composition needs to be decocted before taking, which increases the complexity of drug use.

[0005] Therefore, there is an urgent need for a method for effectively extracting the effective therapeutic ingredients in pediatric abdominal pain grass and preparing them into external preparations that directly act on the lesions and reduce the pain of taking the medicine. Summary of the Invention

[0006] The purpose of the present invention is to provide an enzyme-responsive hydrogel for the treatment of inflammatory bowel disease, and its preparation method and application, so as to solve the problems of insufficient extraction of effective therapeutic ingredients in the above-mentioned pediatric abdominal pain grass and inconvenient direct use of medicinal preparations.

[0007] To achieve the above objectives, the first aspect of the present invention provides an enzyme-responsive hydrogel for the treatment of inflammatory bowel disease. Calculated by weight, the enzyme-responsive hydrogel comprises 13-18% of the effective part of Herba Epimedii Herba, 75-85% of the enzyme-responsive gel matrix, 0.5-1% of purified water, 2-4% of a transdermal absorption enhancer, 0.5-1% of a solubilizer, and 0-0.1% of a preservative.

[0008] Preferably, the enzyme-responsive gel matrix is ​​triglycerol monostearate, the transdermal absorption enhancer is at least one of azone, oleic acid, and propylene glycol, the solubilizer is at least one of Tween-80, propylene glycol, polyethylene glycol, and Span, and the preservative is at least one of ethylparaben, phenoxyethanol, and hexylene glycol.

[0009] Preferably, the preparation method of the effective part of the Herba Lycopodii Herba is as follows:

[0010] (1) Extracting the herb of pediatric abdominal pain with ethanol reflux extraction to obtain an extract, and concentrating the extract under reduced pressure to obtain a medicinal solution;

[0011] (2) The medicinal solution is purified by using D101 macroporous resin, and after adsorption saturation, it is eluted with purified water and ethanol in sequence to obtain an eluate, and the eluate is concentrated to a thick paste under reduced pressure and freeze-dried to obtain the effective part of the pediatric abdominal pain grass.

[0012] Preferably, in step (1), the herb is reflux-extracted with 50-70% ethanol for more than two times, each time for 0.5-1.5 hours, filtered and combined to obtain an extract; the concentration of the extract is 0.1-0.3 g / mL.

[0013] Preferably, in step (2), the sample volume of the drug solution is 1 to 3 BV. After adsorption saturation, it is eluted with 2 to 4 BV of purified water, then eluted with 1 to 3 BV of 10% ethanol, then eluted with 1 to 3 BV of 20% ethanol, and finally eluted with 2 to 4 BV of 40% ethanol. The eluate with 40% ethanol is collected and concentrated under reduced pressure to obtain a thick paste.

[0014] Preferably, in step (2), the effective fraction of Herba Lycopodii var. chebula comprises swertiamarin and gentiopicroside, and the total amount of swertiamarin and gentiopicroside is not less than 50%.

[0015] A second aspect of the present invention provides a method for preparing an enzyme-responsive hydrogel for treating inflammatory bowel disease, comprising the following steps:

[0016] S1: mixing the enzyme-responsive gel matrix, purified water, and a solubilizing agent, and heating and dissolving the mixture to obtain a mixture;

[0017] S2: adding the effective part of Herba Lycopodii Herba, a transdermal absorption enhancer and a preservative to the mixture, stirring, standing and condensing to obtain an enzyme-responsive hydrogel.

[0018] Preferably, in step S1, the heating temperature is 60-80°C.

[0019] A third aspect of the present invention provides an application of an enzyme-responsive hydrogel for treating inflammatory bowel disease, and an application of the enzyme-responsive hydrogel in preparing a medicament for treating inflammatory bowel disease.

[0020] Preferably, the enzyme-responsive hydrogel is applied to the Shenque acupoint, and the effective part of the pediatric abdominal pain grass in the enzyme-responsive hydrogel is transdermally administered through the Shenque acupoint.

[0021] Therefore, the present invention adopts the above-mentioned enzyme-responsive hydrogel for treating inflammatory bowel disease and its preparation method and application, which has the following beneficial effects:

[0022] (1) The present invention uses ethanol of a specific concentration to reflux extract the herb of pediatric abdominal pain and uses a macroporous resin for purification, which can effectively extract and enrich the active ingredients (such as swertiamarin and gentiopicroside) from the herb of pediatric abdominal pain, ensuring that the total amount of these active ingredients in the final product is not less than 50%, thereby improving the therapeutic effect.

[0023] (2) The hydrogel of the present invention is enzyme-responsive and can release drugs in the presence of specific enzymes. This property enables it to act more precisely on the lesion site in the treatment of inflammatory bowel disease, reduce systemic side effects, and improve the safety and effectiveness of treatment.

[0024] (3) The present invention selects the Shenque acupoint as the transdermal drug delivery site, utilizing the unique connection between acupoints and internal organs to help the drug act more directly on the intestines, enhancing local therapeutic effects. At the same time, this method avoids the problems of first-pass effect and gastrointestinal discomfort that may be caused by oral administration.

[0025] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a scanning electron microscope (SEM) image of the enzyme-responsive hydrogel prepared in Example 1;

[0027] Figure 2The particle size and Zeta potential of the enzyme-responsive hydrogel prepared in Example 1;

[0028] Figure 3 This is a graph showing viscosity versus shear rate scanning of the enzyme-responsive hydrogel prepared in Example 1;

[0029] Figure 4 Graph showing the modulus of the enzyme-responsive hydrogel prepared in Example 1 versus amplitude sweep (frequency 1 Hz, range 0.1-1000%);

[0030] Figure 5 This is a diagram of the enzyme-responsive hydrogel structure recovery experiment prepared in Example 1;

[0031] Figure 6 Cumulative permeation curves per unit area of ​​skin at different parts of the body;

[0032] Figure 7 Cumulative permeation curve diagram for enzyme response mode;

[0033] Figure 8 Figure 2 is the weight change of mice;

[0034] Figure 9 This is the DAI score diagram for mice;

[0035] Figure 10 This is a graph showing the effect of the enzyme-responsive hydrogel prepared in Example 1 on serum inflammatory factors in DSS-induced UC model mice;

[0036] Figure 11 This is the pathological picture of HE staining of intestinal tissue section;

[0037] Figure 12 This is a Masson-stained pathological image of intestinal tissue sections;

[0038] Figure 13 IL-1β immunohistochemistry image (×200);

[0039] Figure 14 IL-6 immunohistochemistry images (×200);

[0040] Figure 15 This is the immunohistochemistry image of TNF-α (×200). DETAILED DESCRIPTION

[0041] The present invention will be further described below. It should be noted that this embodiment is based on the technical solution and provides a detailed implementation method and specific operation process, but the present invention is not limited to this embodiment.

[0042] Example 1

[0043] A method for preparing an enzyme-responsive hydrogel for treating inflammatory bowel disease comprises the following steps:

[0044] S1: The preparation method of the effective part of the herb is as follows:

[0045] Weigh 50 g of Herba Epimedii Herba Epimedii Herba Epimedii, extract with 60% ethanol under heating reflux at 90°C according to a solid-liquid ratio of 1:25, extract twice for 1 h each time, filter, combine the filtrates to obtain an extract, and concentrate the extract under reduced pressure to a medicinal solution equivalent to 0.2 g of the herbal slice per 1 mL;

[0046] The medicinal solution was purified using D101 macroporous resin, with a sample volume of 1BV and adsorption at a volume flow rate of 1mL / min. After adsorption saturation, it was first eluted with 3BV purified water, then with 2BV 10% ethanol, then with 1BV 20% ethanol, and finally with 3BV 40% ethanol. 40% of the eluate was collected, concentrated into a paste under reduced pressure, and freeze-dried for 48 hours to obtain the effective part of the pediatric abdominal pain grass. After testing, the total amount of scutellaria baicalensis and gentiopicroside in the effective part of the pediatric abdominal pain grass was 53%.

[0047] S2: Accurately weigh 100 mg of triglycerol monostearate (TG-18), 1 mg of water, and 1 mg of Tween-80, and heat to 70°C until TG-18 is completely dissolved. Then add 20 mg of the effective part of pediatric abdominal pain grass, 3.8 mg of azone, and 0.12 mg of ethyl paraben, and mix well. After standing and cooling, an enzyme-responsive hydrogel is formed.

[0048] The effective part of the herb of pediatric abdominal pain in the present invention is subjected to single factor and response surface method optimization of the extraction process to obtain the highest content of swedemarin and gentiopicroside. The herb of pediatric abdominal pain extract is purified by macroporous resin through single factor and response surface method to obtain the effective part, and the effective part with the highest content of effective ingredients is obtained, making it more effective.

[0049] Example 2

[0050] The enzyme-responsive hydrogel prepared in Example 1 was characterized.

[0051] (1) Morphological characterization

[0052] Take an appropriate amount of enzyme-responsive hydrogel, freeze-dry it at -80℃ for 48 hours, spray-coat the sample with gold, and observe the morphology of the test sample using a scanning electron microscope (SEM). Figure 1 .

[0053] (2) Particle size and Zeta potential of enzyme-responsive hydrogels

[0054] The prepared enzyme-responsive hydrogel was diluted 10 times and its particle size distribution and Zeta potential were measured using a laser particle size analyzer. The particle size of the enzyme-responsive hydrogel was 77.4±1.20nm and the Zeta potential was -34.1±1.02mV. Figure 2 .

[0055] (3) Rheological properties of enzyme-responsive hydrogels

[0056] Rheology is the property between deformation and flow of a substance under the action of external force, which aims to characterize the quantitative relationship between strain and stress of a substance under the action of external force. For hydrogels, in rheological tests, the parameters that generally need to be tested include storage modulus G' and loss modulus G", which characterize the solid-like behavior and liquid-like behavior of the substance respectively. The G' and G" of the hydrogel sample are measured by dynamic frequency scanning method (0.01% strain). The above parameters can reflect the elasticity and viscosity of the object respectively. Operating parameters: temperature is 25°C, scanning range is 0.1~10Hz. Figure 3 As shown in Figure 2, the viscosity of the enzyme-responsive hydrogel decreases sharply with increasing shear rate. The storage modulus (G') and loss modulus (G"), as shown in Figure 2, Figure 4 As shown in Figure 2, with the increase of strain, the G' and G" curves intersect at a strain of 10%, which is the critical strain value for the gel network to transition to a liquid state. Figure 5 As shown in the figure, in the low strain region, the storage modulus (G') and the loss modulus (G") both maintain constant values. As the strain increases to 100%, both G' and G" decrease significantly, and G' is greater than G", indicating that the enzyme-responsive hydrogel has good self-healing properties.

[0057] Example 3

[0058] The enzyme-responsive hydrogel prepared in Example 1 was subjected to an in vitro transdermal test.

[0059] (1) Test method

[0060] A modified FRANZ diffusion cell was used, the receiving solution was 20% ethanol-normal saline, the receiving cell volume was 10 mL, the inner diameter was 1.44 cm, the temperature of the modified FRANZ diffusion cell was maintained at 37.0±0.1°C, and the stirring speed was 350 r / min. The enzyme-responsive hydrogel was evenly applied to the mouse skin. 2 mL of receiving solution was aspirated at 1, 2, 4, 8, 12, and 24 h, and 2 mL of fresh receiving solution was quickly added. Each time, the bubbles under the skin were expelled. The samples obtained at each time point were filtered through a 0.22 μm filter membrane, and the concentrations of scutellarin and gentiopicroside were determined by HPLC. The cumulative permeation amount of scutellarin and gentiopicroside at each time point was calculated according to the following formula: Cumulative permeation amount per unit area (Q n , μg·cm -2 ).

[0061]

[0062] Where C n is the drug concentration measured at the nth sampling point (μg·mL -1 );C i is the drug concentration measured at the i-th sampling point (μg·mL -1 ); A is the effective transdermal permeation area of ​​the diffusion cell; V is the volume of the receiving fluid; V i is the sampling volume.

[0063] (2) Comparison of transdermal penetration in different parts of mice

[0064] For mouse acupoint skin, sample the area between the upper 3 / 4 and lower 1 / 4 of the line connecting the upper edge of the sternum to the external genitalia, located in the middle of the mouse chest and abdomen. For non-acupoint skin, sample the area 1-2 cm away from the Shenque point (avoiding the meridian line).

[0065] Several Kunming mice were secured and shaved with an electric shaver. The remaining short hair was then removed by applying a depilatory cream to the abdomen. After cervical dislocation, two skin sections were removed from the Shenque acupoint and the area adjacent to the Shenque acupoint (not the acupoint area). The subcutaneous mucosa and adipose tissue were scraped off, and the skin was rinsed with saline until the wash was clear. The skin was then soaked in saline and stored in a refrigerator to serve as spare skin for in vitro transdermal testing.

[0066] Depend on Figure 6 It can be seen that in the in vitro experimental release of enzyme-responsive hydrogel, the cumulative permeability of the abdominal skin located at the Shenque point is greater than that of the abdominal skin at non-acupoints.

[0067] Table 1 Cumulative permeation of swirtiamarin and gentiopicroside in enzyme-responsive hydrogels at different skin sites in mice (x±s, n=3)

[0068]

[0069]

[0070] As shown in Table 1, the cumulative permeation amount of swedemarin and gentiopicroside per unit area of ​​the enzyme-responsive hydrogel in the skin of the Shenque acupoint is 272.12.31±7.71μg / cm 2 , which is 2.82 times that of the enzyme-responsive hydrogel in the effective part of pediatric abdominal pain grass on the abdominal skin other than the Shenque point.

[0071] (3) Enzyme-responsive release

[0072] The cumulative permeation amount of swirtsmarin and gentiopicroside in the enzyme-responsive hydrogel (Qn , μg / cm 2 ).

[0073] Table 2 Cumulative permeation per unit area of ​​enzyme-responsive hydrogels in the presence and absence of enzymes in the release of swedemarin and gentiopicroside (x±s, n=3)

[0074]

[0075] Depend on Figure 7 As shown in Table 2, the cumulative permeation amount of scutellarin and gentiopicroside in the enzyme-responsive hydrogel containing enzyme release was 412.43±3.49μg / cm 2 , 1.73 times the release without enzymes. This suggests that enzymes can promote gel decomposition and accelerate drug release. TG-18 hydrogels are composed of ester bonds. Enzymes such as T. lanuginosus lipase, MMP2, and MMP9 can break down the ester bonds of TG-18 hydrogels through enzymatic hydrolysis, leading to hydrogel erosion and localized drug release.

[0076] Example 4

[0077] Anti-inflammatory therapeutic effect of the enzyme-responsive hydrogel prepared in Example 1 on UC mice.

[0078] (1) Experimental animals

[0079] SPF C57BL / 6 male mice, 8 weeks old and weighing 20–22 g, were purchased from Besford (Beijing) Biotechnology Co., Ltd. Animal Certification No. 110324241105418245, License No. SCXK (Beijing) 2024-0001, and Ethics Approval No. XMSQ202409019. They were maintained at a temperature of 22–24°C and a relative humidity of 40%–60%. Adaptive feeding was performed for one week.

[0080] (2) Animal grouping and drug administration

[0081] After adaptive feeding, C57BL / 6 mice were randomly divided into a blank group, a model group, a positive drug group, a drug-treated group (at the Shenque point), and a drug-treated group (not at the Shenque point), with 8 mice in each group. The blank group received free food, while the remaining groups were gavaged with 3% (w / v) DSS for model establishment. Model establishment and drug administration were performed simultaneously for 7 consecutive days, and samples were collected 24 hours after drug administration on the 7th day.

[0082] Dosing: The day before the experiment, hair of approximately 2 cm × 2 cm was removed from the abdomen using electric clippers. A small amount of depilatory cream was then used to remove the remaining small hair on the mouse abdomen. After depilation, the prepared blank hydrogel and enzyme-responsive hydrogel were administered percutaneously once daily. The positive drug group was given 7.8 mg / 20 g SASP (sulfasalazine) by gavage. After application, the mice were wrapped with medical gauze and fixed with non-irritating tape.

[0083] Table 3 Animal experimental groups and dosages

[0084]

[0085]

[0086] (3) Model evaluation indicators

[0087] During the modeling period, the weight and activity level of the mice were monitored, and their condition, including hair, coat color, and whether symptoms such as blood in the stool and rectal prolapse occurred, were observed. The mice were scored based on their daily stool consistency and blood in the stool, and the scores of weight change, stool consistency, and blood in the stool were added together to obtain the disease activity index (DAI) score. The DAI was evaluated according to the established standards, and the score was obtained by adding the fecal occult blood degree score and the weight loss rate score. The final score was between 0 and 8 points, thereby obtaining the average DAI score for each group of mice, which can be used to roughly evaluate the disease activity status (clinical manifestations) of each group of mice. Disease activity index (DAI) = (body weight loss rate score + fecal characteristics score + occult blood degree score) / 3, weight loss rate (%) = (weight before the experiment - weight after modeling) / weight before the experiment × 100%.

[0088] Table 4 Disease Activity Index Score

[0089]

[0090] (4) Elisa detection of TNF-α, IL-6, and IL-1β expression levels

[0091] The enzyme-linked immunosorbent assay (ELISA) method was used to determine the levels of inflammatory factors TNF-α, IL-6, and IL-1β according to the kit instructions.

[0092] (5) HE staining of intestinal tissue sections

[0093] Colon tissue fixed with paraformaldehyde was dehydrated in 70%, 80%, 95%, and 100% ethanol, then cleared in xylene for 30 minutes and embedded in paraffin blocks. Groups were labeled on the paraffin blocks, and tissue sections approximately 4 μm thick were cut and placed in a preheated water bath. The tissue was removed using a cationic anti-dewaxing slide, dried, and then labeled on the slides for detailed information about each sample. Prepared tissue sections were dewaxed in xylene I and II, then hydrated in 100%, 95%, 80%, and 70% ethanol solutions, followed by rinsing in running water. Sections were stained in hematoxylin solution and rinsed in tap water. Differentiation was performed in 1% hydrochloric acid-alcohol for 1 minute, followed by immediate rinsing in tap water and then immersion in tap water to restore the blueing. Stain the sections in eosin solution for 2 minutes, rinse with tap water, dehydrate the sections in 70%, 80%, 95%, and 100% ethanol, and then immerse in xylene solution for 2 minutes to clear the sections. Finally, seal the sections with neutral gum and observe the staining results under an optical microscope.

[0094] (6) Masson staining of intestinal tissue sections

[0095] Embed the sections using conventional paraffin wax methods and slice (3 μm thickness). After dewaxing and hydration, the sections were placed in Bouin's solution and mordanted in a 37°C incubator for 2 hours. The sections were then rinsed with running water until the yellow color on the surface of the sections disappeared. Next, the sections were stained with lapis lazuli blue for 2–3 minutes, followed by Mayer's hematoxylin for 2–3 minutes. After differentiation in acidic ethanol solution for a few seconds, the sections were rinsed with running water for 10 minutes. Next, the sections were stained with Ponceau fuchsin for 10 minutes, followed by treatment with phosphomolybdic acid solution for approximately 10 minutes. Following staining, the sections were stained with aniline blue for 5 minutes and treated with a weak acid solution for 2 minutes. Rapid dehydration was performed using 95% ethanol and then dehydrated three times with anhydrous ethanol for 5–10 seconds each. Finally, the sections were cleared with xylene three times for 1–2 minutes each and mounted with neutral gum.

[0096] (7) IHC (immunohistochemistry) method to detect the expression of TNF-a, IL-6, and IL-1β in mouse colon

[0097] Colon tissue sections were dewaxed and dehydrated, followed by antigen retrieval, endogenous peroxidase blocking, and serum blocking. They were then incubated with primary and secondary antibodies. After rinsing, DAB staining and nuclear staining were performed. Finally, sections were dehydrated and mounted, observed under a microscope, and photographed. Immunohistochemical staining for TNF-α, IL-6, and IL-1β was analyzed.

[0098] (8) Statistical analysis

[0099] Statistical analysis was performed using SPSS 26.0 software. One-way analysis of variance was used to compare experimental data between groups, and t-tests were used for pairwise comparisons. Data are expressed as mean ± standard deviation. P < 0.05 indicated a significant difference.

[0100] (9) Test results

[0101] ①Effects of enzyme-responsive hydrogel on body weight and disease activity index of UC mice

[0102] like Figure 8 As shown in the figure, starting from the third day after modeling, the weight of mice in each group continued to decrease, and the weight loss in the model group was the most obvious. After the sixth day, the weight loss trend of mice in each group slowed down. At the end of the experiment, the weight loss of mice in the model group was the most obvious compared with the drug group (via Shenque point), drug group (not via Shenque point), and positive drug group. Figure 9 As shown in the figure, before sacrifice, mice in each group were assessed for DAI scores based on blood in stool, stool characteristics, and weight loss. The results showed that compared with the normal group, the model group had decreased weight and increased DAI scores (P < 0.05). Compared with the model group, mice in the drug-treated group (via the Shenque acupoint), drug-treated group (not via the Shenque acupoint), and positive drug group had increased weight and decreased DAI scores (P < 0.05). Overall, the improvement of UC symptoms in mice was as follows: positive drug group > drug-treated group (via the Shenque acupoint) > drug-treated group (not via the Shenque acupoint). The drug-treated group (via the Shenque acupoint) had a greater effect than the drug-treated group (not via the Shenque acupoint), which may be related to the structural and electrophysiological characteristics of the skin at the Shenque acupoint. The skin at the Shenque acupoint in mice is thinner and has more hair follicles than the skin at non-acupoint sites, which facilitates transdermal drug absorption. Furthermore, both humans and animals experience the phenomenon of low-impedance along the meridian: the impedance at acupoints along the meridian is lower than at the sides, resulting in higher drug delivery efficiency at low-impedance skin.

[0103] ②Effects of enzyme-responsive hydrogel on the levels of TNF-a, IL-6, and IL-1β in the serum of UC mice

[0104] like Figure 10 As shown in the figure, the results of ELISA detection of serum inflammatory factors showed that compared with the normal group, the expression levels of TNF-a, IL-6, and IL-1β in the model group were increased (P<0.01). Compared with the model group, the expression levels of TNF-a, IL-6, and IL-1β in the drug group (via Shenque point), drug group (not via Shenque point), and positive drug group were decreased (P<0.01). Overall, the effect of reducing the levels of TNF-a, IL-6, and IL-1β in UC mice showed the following order: positive drug group > drug group (via Shenque point) > drug group (not via Shenque point).

[0105] ③HE staining of intestinal tissue sections

[0106] The microscopic morphology of the colon of each group of mice was observed by HE staining and scored. Figure 11 As shown in the figure. The colons of mice in the blank control group displayed intact structures, with neatly arranged goblet cells, crypts, and intestinal glands, and no significant edema. Compared with the blank control group, mice in the model group showed loss of colonic epithelial structure, loose goblet cells, destroyed crypts, thickened mucosal layer, and inflammatory cell infiltration in the mucosa and submucosa. The histopathological injury score was the highest, significantly different from the blank control group (P < 0.01). Compared with the model group, the drug group (via Shenque point), drug group (not via Shenque point), and positive drug group all alleviated these symptoms. Overall, the degree of reduction in the microscopic morphology of the UC mouse colon showed the following: positive drug group > drug group (via Shenque point) > drug group (not via Shenque point).

[0107] ④Masson staining of intestinal tissue sections

[0108] Masson staining can make the collagen fibers deposited in the colon appear blue. Figure 12 As shown, the colon tissue of mice in the normal group was neatly arranged, with no fibrotic areas in the mucosa and submucosa. Compared with the normal group, the colon tissue of mice in the model group showed numerous blue-stained areas in the mucosa and submucosa, with collagen fiber deposition and proliferation (P<0.01). Compared with the model group, the collagen fiber deposition in the colon tissue of mice in the drug group (via Shenque acupoint), drug group (not via Shenque acupoint), and positive drug group was reduced (P<0.05). Overall, the degree of collagen fiber reduction in the colon of UC mice showed the following pattern: positive drug group > drug group (via Shenque acupoint) > drug group (not via Shenque acupoint).

[0109] ⑤IHC (immunohistochemistry) method was used to detect the expression of IL-1β, IL-6 and TNF-a in the mouse colon

[0110] The results of immunohistochemical staining for IL-1β are shown in Figure 13 , only a small amount of expression was found in the colon mucosa and submucosal tissues of the mice in the normal group, and the small amount of expression was located in scattered inflammatory cells; compared with the normal group, the positive areas of the diseased colon of the mice in the DSS group were mainly located in the cytoplasm and cell membrane of a large number of lymphocytes, monocytes, neutrophils and other cells in the submucosal layer and next to the blood vessels. Brown or brown particles were observed under the microscope, and the expression of IL-1β was significantly increased, and the difference was statistically significant (*P<0.05). Compared with the DSS group, the expression intensity of IL-1β was significantly weakened in the drug group (administered via Shenque point), the drug group (not administered via Shenque point), and the positive drug group, and the difference was statistically significant ( # P<0.05). In general, the degree of reduction in IL-1β expression in the colon of UC mice showed: positive drug group > drug administration group (via Shenque acupoint) > drug administration group (not via Shenque acupoint).

[0111] Immunohistochemical staining results for IL-6 are shown in Figure 14 In the colon mucosal tissue of mice in the normal group, a small amount of IL-6 was expressed in the epithelial cells and scattered inflammatory cells in the submucosal tissue; compared with the normal group, a large number of inflammatory cells were found in the epithelial cells and submucosal layer of the diseased colon mucosa of mice in the DSS group, and many brown or brown particles were observed under the microscope. The expression of IL-6 was significantly increased, and the difference was statistically significant (*P<0.05). Compared with the DSS group, the IL-6 expression intensity of the drug group (administered via Shenque point), the drug group (not administered via Shenque point), and the positive drug group was significantly weakened, and the difference was statistically significant ( # P<0.05). In general, the degree of reduction in IL-6 expression in the colon of UC mice showed: positive drug group > drug administration group (via Shenque acupoint) > drug administration group (not via Shenque acupoint).

[0112] Immunohistochemical staining results for TNF-α Figure 15 In the colon mucosal tissue of mice in the normal group, a small amount of TNF-α was expressed in scattered mucosal epithelial cells and inflammatory cells. Compared with the normal group, the expression of TNF-α in the cytoplasm and cell membrane of the epithelial cells and a large number of inflammatory cells in the lesion colon of mice in the DSS group was significantly enhanced. Microscopic observation showed diffuse brown or brown granular cytoplasm staining, and scattered positive cells were also seen in the mucosal lamina propria and lymph nodules. The difference was statistically significant (*P<0.05). Compared with the DSS group, the expression intensity of TNF-α in the drug group (drug administration via Shenque point), the drug group (not drug administration via Shenque point), and the positive drug group was significantly weakened. The difference was statistically significant ( # P<0.05). In general, the degree of reduction in TNF-α expression intensity in the colon of UC mice showed: positive drug group > drug administration group (via Shenque acupoint) > drug administration group (not via Shenque acupoint).

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An enzyme-responsive hydrogel for treating inflammatory bowel disease, characterized in that: Calculated by weight percentage, the enzyme-responsive hydrogel includes 13-18% of the effective part of the pediatric abdominal pain grass, 75-85% of the enzyme-responsive gel matrix, 0.5-1% of purified water, 2-4% of a transdermal absorption enhancer, 0.5-1% of a solubilizer, and 0-0.1% of a preservative.

2. The enzyme-responsive hydrogel for treating inflammatory bowel disease according to claim 1, characterized in that: The enzyme-responsive gel matrix is ​​triglycerol monostearate, the transdermal absorption enhancer is at least one of azone, oleic acid, and propylene glycol, the solubilizer is at least one of Tween-80, propylene glycol, polyethylene glycol, and Span, and the preservative is at least one of ethylparaben, phenoxyethanol, and hexylene glycol.

3. The enzyme-responsive hydrogel for treating inflammatory bowel disease according to claim 1, characterized in that: The preparation method of the effective part of the Herba Lycopodii Herba is as follows: (1) Take Herba Epimedii Herba Epimedii, extract it with ethanol under reflux to obtain an extract, and concentrate the extract under reduced pressure to obtain a medicinal solution; (2) The medicinal solution is purified using D101 macroporous resin. After adsorption saturation, it is eluted with purified water and ethanol in sequence to obtain an eluate. The eluate is concentrated under reduced pressure to a thick paste and freeze-dried to obtain the effective part of the pediatric abdominal pain grass.

4. The enzyme-responsive hydrogel for treating inflammatory bowel disease according to claim 3, characterized in that: In step (1), the herb is extracted with 50-70% ethanol under reflux for more than 2 times, each time for 0.5-1.5 hours, filtered and combined to obtain an extract; The concentration of the drug solution is 0.1~0.3g / mL.

5. The enzyme-responsive hydrogel for treating inflammatory bowel disease according to claim 3, characterized in that: In step (2), the loading volume of the drug solution is 1~3BV. After adsorption saturation, it is eluted with 2~4BV of purified water, then eluted with 1~3BV of 10% ethanol, then eluted with 1~3BV of 20% ethanol, and finally eluted with 2~4BV of 40% ethanol. The eluate with 40% ethanol is collected and concentrated under reduced pressure to obtain a thick paste.

6. The enzyme-responsive hydrogel for treating inflammatory bowel disease according to claim 3, characterized in that: In step (2), the effective parts of the pediatric abdominal pain grass include swertiamarin and gentiopicroside, and the total amount of swertiamarin and gentiopicroside is not less than 50%.

7. The method for preparing an enzyme-responsive hydrogel for treating inflammatory bowel disease according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1: mixing the enzyme-responsive gel matrix, purified water, and a solubilizing agent, and heating and dissolving the mixture to obtain a mixture; S2: adding the effective part of Herba Lycopodii Herba, a transdermal absorption enhancer and a preservative to the mixture, stirring, standing and condensing to obtain an enzyme-responsive hydrogel.

8. The method for preparing an enzyme-responsive hydrogel for treating inflammatory bowel disease according to claim 7, characterized in that: In step S1, the heating temperature is 60-80°C.

9. Use of an enzyme-responsive hydrogel for treating inflammatory bowel disease according to any one of claims 1 to 6, characterized in that: Application of enzyme-responsive hydrogels in the preparation of therapeutic agents for inflammatory bowel disease.

10. The use of an enzyme-responsive hydrogel for treating inflammatory bowel disease according to claim 9, characterized in that: The enzyme-responsive hydrogel is applied to the Shenque acupoint, and the effective part of the pediatric abdominal pain grass in the enzyme-responsive hydrogel is transdermally administered through the Shenque acupoint.

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