Ursodeoxycholic acid-hyaluronic acid nano-emulsion and application thereof in treatment of inflammatory bowel disease

Abstract

The invention discloses an ursodesoxycholic acid-hyaluronic acid nano-emulsion and an application of the ursodesoxycholic acid-hyaluronic acid nano-emulsion in treatment of inflammatory bowel diseases. The nano-emulsion is high in stability, and has excellent antioxidant, anti-inflammatory and active oxygen scavenging capabilities: through specific binding and electrostatic interaction of hyaluronic acid and a CD44 receptor, the nano-emulsion is accurately delivered to an inflammatory site, mucosal barrier repair is promoted, and intestinal flora and an inflammatory microenvironment are regulated; the FXR pathway can be activated, the NF-kappa B signal pathway can be inhibited, the immune microenvironment can be regulated, the generation of proinflammatory factors can be reduced, and the bile acid metabolism can be balanced. The compound has a good curative effect in a DSS-induced colitis mouse model, can reduce lipopolysaccharide entering blood, reduce liver inflammation and regulate liver metabolism, and provides an important theoretical basis for IBD treatment based on intestine-liver regulation.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation technology, specifically relating to an ursodeoxycholic acid-hyaluronic acid nanoemulsion, its preparation method, and its application in the treatment of inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a complex intestinal disease mediated by multiple factors. Its typical pathological features include intestinal mucosal damage, disruption of the epithelial barrier function, abnormal immune response, and gut microbiota dysbiosis. These factors interact and promote each other, jointly driving disease progression and often accompanied by extraintestinal organ damage. The intestine and liver form a close bidirectional regulatory pathway, namely the gut-hepatic axis, through the bile duct, portal vein, and systemic circulation: the liver can secrete bioactive substances such as bile acids into the intestine via the bile duct, while small and large molecules from the intestine can also act on the liver via the portal vein. The signaling interaction between gut-derived and hepatic-derived factors constitutes a complex regulatory network and has become a key link in elucidating the pathogenesis of IBD. Therefore, targeted regulation of gut-hepatic axis homeostasis holds promise for providing a novel treatment strategy for inflammatory bowel disease.

[0003] Bile acids are the end products of cholesterol metabolism in the liver. As key signaling molecules, they participate in gut-hepatic axis signaling, regulating not only lipid absorption and metabolism but also gut microbiota structure, maintaining intestinal barrier function, and modulating the intestinal mucosal immune microenvironment through bile acid receptors (mainly FXR). Bile acid metabolism disorders are common in IBD patients, and damaged intestinal epithelium further reduces bile acid absorption efficiency and FXR receptor activity. Studies have shown that obeticholic acid (OCA) can repair the mucosal barrier and alleviate colitis by targeting the hepatic FXR-CYP8B1 signaling pathway; conjugated bile acids such as taurocholic acid (TCA) can exert anti-inflammatory effects by strengthening the intestinal barrier, promoting mucus secretion, and activating the FXR / TGR5 pathway. These studies confirm that gut-hepatic axis-based bile acid metabolism disorders play a crucial role in the development and progression of colitis, providing an important theoretical basis for reshaping bile acid metabolism and treating IBD.

[0004] Ursodeoxycholic acid (UDCA) is a commonly used clinical treatment for hepatobiliary diseases, regulating bile acid metabolism and reducing hepatocellular damage. Studies have shown that UDCA can indirectly activate intestinal FXR receptors by remodeling the gut microbiota structure, optimizing secondary bile acid production, promoting Akkermania colonization, inhibiting inflammatory and abnormal proliferation pathways, and reducing bacterial translocation and endotoxin (LPS) production, thereby playing a role in the prevention of colitis. However, UDCA has significant limitations in clinical application: long-term use easily leads to drug resistance, and it has poor water solubility and low intestinal absorption efficiency. Therefore, targeted modification of UDCA to improve its targeted enrichment ability at inflammatory sites is expected to reduce the dosage and frequency of administration, achieving better therapeutic effects with lower effective doses. Summary of the Invention

[0005] To address the problems of poor water solubility, low absorption efficiency, and lack of inflammatory targeting in existing technologies, as well as the accompanying intestinal barrier damage, bile acid metabolism disorders, and gut microbiota dysbiosis in inflammatory bowel disease (IBD), which further induces liver damage, this invention aims to provide an ursodeoxycholic acid-hyaluronic acid nanoemulsion, its preparation method, and its application in IBD. This nanoemulsion can efficiently scavenge reactive oxygen species at sites of intestinal inflammation, regulate gut microbiota homeostasis, repair the intestinal mucosal barrier, and improve liver inflammation and damage mediated by gut microbiota dysbiosis while treating ulcerative colitis. Furthermore, by regulating bile acid and fatty acid metabolism and reducing lipopolysaccharide (LPS) entry into the bloodstream, it provides a safe, efficient, and targeted treatment option for IBD and its gut-hepatic axis-related complications.

[0006] To solve the problems of the prior art, the present invention adopts the following technical solution: This invention first provides a method for preparing ursodeoxycholic acid-hyaluronic acid nanoemulsion, characterized by: covalently linking hyaluronic acid and ursodeoxycholic acid through a two-step amidation reaction to construct an ursodeoxycholic acid-hyaluronic acid coupling compound with targeting capabilities to inflammatory sites; then using this coupling compound as an emulsification carrier, encapsulating lipid-soluble antioxidant oils through an oil-in-water emulsification technique to form a stable, targeted ursodeoxycholic acid-hyaluronic acid nanoemulsion with both antioxidant and anti-inflammatory functions. This nanoemulsion can precisely accumulate at sites of intestinal inflammation, regulate intestinal flora homeostasis, repair the intestinal mucosal barrier, and synergistically regulate bile acid and fatty acid metabolism with ursodeoxycholic acid, achieving effective treatment for inflammatory bowel disease.

[0007] The preparation method of the ursodeoxycholic acid-hyaluronic acid nanoemulsion of the present invention specifically includes the following steps: 1) Hyaluronic acid was dissolved in deionized water, and activators 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added and stirred for 30-40 min to activate the solution. Then, ethylenediamine was added to carry out the first-step amidation reaction (stirred at room temperature for 24-36 h). The reaction solution was dialyzed and lyophilized to obtain hyaluronic acid-ethylenediamine. 2) Hyaluronic acid-ethylenediamine was dissolved in deionized water to obtain solution 1; ursodeoxycholic acid was dissolved in N,N-dimethylformamide (DMF), and EDC and NHS were added and stirred to activate the solution to obtain solution 2; solution 1 was slowly added to solution 2 to carry out the second amidation reaction (stirred at room temperature for 12 h ~ 24 h), and the reaction solution was dialyzed and lyophilized to obtain ursodeoxycholic acid-hyaluronic acid coupling compound; 3) Dissolve the ursodeoxycholic acid-hyaluronic acid coupling compound in deionized water, add lipid-soluble antioxidant oil, and stir using a homogenizer to obtain ursodeoxycholic acid-hyaluronic acid nanoemulsion.

[0008] The fat-soluble antioxidant oils include, but are not limited to, one or more of the following: Ganoderma lucidum spore oil, vitamin E, rice bran oil, and fish oil.

[0009] Preferably, in step 1), the molar ratio of hyaluronic acid, ethylenediamine, NHS, and EDC is 1-2:1-3:1-2:1-2.

[0010] Preferably, in step 2), the molar mass ratio of ursodeoxycholic acid, hyaluronic acid-ethylenediamine, NHS and EDC is 1-2:1-3:1-2:1-2.

[0011] Preferably, in steps 1) and 2), the molecular weight cutoff of the dialysis bag used for dialysis is 10 kDA-20 kDA.

[0012] Preferably, in step 3), the ratio of ursodeoxycholic acid-hyaluronic acid coupling compound, deionized water and fat-soluble antioxidant oil is 20-80 mg: 20-100 mL: 5-20 mL.

[0013] Preferably, in step 3), the homogenizer rotates at 10,000 to 12,000 rpm and the mixing time is 15 to 40 minutes.

[0014] The ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in this invention can be used to prepare drugs for the prevention and / or treatment of inflammatory bowel disease. Its mechanism of action is as follows: the nanoemulsion can precisely target and deliver to the site of intestinal inflammation through the specific binding and electrostatic interaction between hyaluronic acid and CD44 receptors; under the action of intestinal microorganisms and hyaluronidase, it slowly releases lipid-soluble antioxidant oils, effectively scavenging reactive oxygen species at the site of inflammation and protecting intestinal epithelial cells; simultaneously, it can activate FXR receptors, inhibit the NF-κB signaling pathway, reduce the release of pro-inflammatory factors, promote macrophage polarization towards the M2 phenotype, and improve the inflammatory microenvironment. Furthermore, this nanoemulsion can regulate the structure of the intestinal flora, increase the level of short-chain fatty acids, restore the expression of intestinal tight junction proteins, repair the intestinal barrier, reduce lipopolysaccharide (LPS) entry into the bloodstream, thereby alleviating liver inflammation and damage; and through the FXR pathway, it promotes bile acid reabsorption and hepatic bile acid metabolism reprogramming, ultimately achieving the goal of reshaping the gut-hepatic axis homeostasis and treating inflammatory bowel disease.

[0015] Compared with existing technologies, the beneficial effects of this invention are reflected in: 1. This invention constructs an ursodeoxycholic acid-hyaluronic acid targeting carrier through a two-step amidation reaction. By leveraging the specific binding of hyaluronic acid to the CD44 receptor highly expressed at the site of inflammation, it can achieve active targeted enrichment of the intestinal inflammatory site and significantly increase the local concentration of the drug at the lesion site.

[0016] 2. The ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in this invention is loaded with Ganoderma lucidum spore oil, which is rich in active ingredients such as Ganoderma lucidum triterpenes. It has excellent antioxidant and immunomodulatory capabilities, can effectively remove excess reactive oxygen species (ROS) from inflamed sites, and reduce oxidative stress damage.

[0017] 3. The components of the nanoemulsion of the present invention exert a synergistic therapeutic effect, which can reshape the bile acid metabolism homeostasis of the gut-liver axis and significantly alleviate colitis symptoms. It shows good therapeutic effects in both preventive and therapeutic colitis models induced by DSS (sodium dextran sulfate). It can improve liver bile acid metabolism disorder by regulating intestinal bile acid metabolism and effectively reduce liver damage secondary to intestinal inflammation. Attached Figure Description

[0018] Figure 1 The ursodeoxycholic acid-hyaluronic acid prepared in Example 1 ( Figure 1 (a) and ursodeoxycholic acid-hyaluronic acid nanoemulsion ( Figure 1 Transmission electron microscopy (TEM) image of (b) in the image.

[0019] Figure 2 The image shows the zeta potential diagrams of the ursodeoxycholic acid-hyaluronic acid and ursodeoxycholic acid-hyaluronic acid nanoemulsions prepared in Example 1. The inset shows the ursodeoxycholic acid-hyaluronic acid nanoemulsions after emulsification of Ganoderma lucidum spore oil and ursodeoxycholic acid-hyaluronic acid.

[0020] Figure 3 The following are the verification diagrams of the CD44 targeting ability of the ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in Example 1: (a) verification of high expression of CD44 receptor in RAW264.7 cells; (b) verification of the CD44 targeting ability of the nanoemulsion.

[0021] Figure 4 This is a graph demonstrating the ability of the ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in Example 1 to induce ulcerative colitis via targeted DSS.

[0022] Figure 5 ABTS is the ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in Example 1. + Antioxidant performance test chart.

[0023] Figure 6 The following figures demonstrate the ability of the ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in Example 1 to scavenge ROS and protect cells: (a) shows the staining results of DCFH-DA on intracellular ROS; (b) shows the results of cell live / dead fluorescence staining.

[0024] Figure 7The results of the test on the preventive and therapeutic effects of the ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in Example 1 on ulcerative colitis are as follows: (a) is the curve of mouse weight change; (b) is the statistical graph of colon length; (c) is a colon photograph.

[0025] Figure 8 The results of the delayed treatment performance test of the ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in Example 1 on ulcerative colitis are as follows: (a) is the curve of mouse body weight change; (b) is the colon length statistics; (c) is the colon photograph.

[0026] Figure 9 The following figures illustrate the therapeutic effects of the ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in Example 1 on the liver: (a) FITC-glucan assay to assess intestinal permeability; (b) serum LPS level; (c) serum total bile acid level; (d) serum total cholesterol (TC) level; (e) serum triglyceride (TG) level; and (f) serum levels of liver function-related biochemical markers (AST, ALP, LDH, ALT). Detailed Implementation

[0027] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0028] Example 1 In this embodiment, the ursodeoxycholic acid-hyaluronic acid nanoemulsion was prepared according to the following steps: Step 1: First, accurately weigh 1 mmol of hyaluronic acid and dissolve it in 50 mL of deionized water, stirring until completely dissolved to obtain an aqueous hyaluronic acid solution. Then, add 1.2 mmol of EDC and 1.2 mmol of NHS to this aqueous solution and activate by stirring at room temperature for 30 min. Next, slowly add 1.5 mmol of ethylenediamine (EDA) and continue stirring at room temperature for 24 h. After the reaction is complete, place the reaction solution in a 14 kDa dialysis bag and dialyze for 2 days to remove unreacted EDC, NHS, and excess EDA. After dialysis, freeze-dry the resulting solution for 3 days to obtain hyaluronic acid-ethylenediamine (HA-EDA) solid powder.

[0029] Step 2: Take 1.5 mmol of the prepared hyaluronic acid-ethylenediamine solid powder and dissolve it in 50 mL of deionized water. Stir until completely dissolved to obtain solution 1. Accurately weigh 1 mmol of ursodeoxycholic acid (UDCA) and dissolve it in 10 mL of LDM. Stir until completely dissolved. Add 1.2 mmol of EDC and 1.2 mmol of NHS to the solution and stir to activate the reaction for 30 min to obtain solution 2. Slowly add solution 1 to solution 2 dropwise. After the addition is complete, continue stirring at room temperature for 12 h. After the reaction is complete, place the reaction solution in a 14 kDa dialysis bag and dialyze for 2 days to remove unreacted EDC, NHS, and excess UDCA. After dialysis, freeze-dry the resulting solution for 3 days to obtain ursodeoxycholic acid-hyaluronic acid coupling compound solid powder (denoted as HADA).

[0030] Step 3: Weigh 30 mg of the ursodeoxycholic acid-hyaluronic acid coupling compound prepared above, dissolve it in 30 mL of deionized water, and stir until completely dissolved to obtain an aqueous solution of the coupling compound; then, slowly add 5 mL of Ganoderma lucidum spore oil (GLSO) to the above aqueous solution of the coupling compound under the action of a homogenizer at a speed of 12000 rpm, and continue to stir and emulsify to finally obtain ursodeoxycholic acid-hyaluronic acid nanoemulsion (denoted as GLSO@HADA).

[0031] The structure of the ursodeoxycholic acid-hyaluronic acid nanoemulsion prepared in this embodiment was characterized, and the results are as follows: Figure 1 As shown in the transmission electron microscopy (TEM) images, both the ursodeoxycholic acid-hyaluronic acid nanoparticles (HADA) and the ursodeoxycholic acid-hyaluronic acid nanoemulsion (GLSO@HADA) obtained in this embodiment exhibit uniform particle size distribution, regular morphology, and good dispersibility. Figure 2 As shown in the Zeta potential diagram, the detection potential of the nanoemulsion is -11.59±0.81mV, indicating that the ursodeoxycholic acid-hyaluronic acid nanoemulsion has been successfully synthesized and has a negatively charged surface. It can be targeted and enriched in the positively charged inflamed colon through electrostatic interaction, providing structural support for subsequent targeted therapy of inflammatory bowel disease.

[0032] Example 2 This embodiment evaluates the targeted binding ability of the ursodeoxycholic acid-hyaluronic acid nanoemulsion (GLSO@HADA) prepared in Example 1 to the CD44 receptor through cell experiments. Specifically, it includes two parts: verification of high expression of CD44 receptor on inflammatory cells and verification of the targeted uptake ability of nanomaterials. The operation is as follows: I. Verification of high CD44 receptor expression in LPS-stimulated RAW264.7 cells RAW264.7 cells were added at a concentration of 1×10⁻⁶ mL per well. 6Cells were seeded at a density of [number] cells / mL in confocal culture dishes and incubated at 37°C for 24 h. Subsequently, lipopolysaccharide (LPS, final concentration 1 µg / mL) was added for 12 h to construct an inflammatory cell model. Cells without LPS treatment served as a blank control group. After stimulation, cells were subjected to immunofluorescence staining as follows: (1) Discard the culture medium, wash the cells with pre-cooled PBS, add 4% paraformaldehyde to fix at room temperature for 30 min, and then permeate the cell membrane with 0.1% Triton X-100 at room temperature for 20 min; (2) Block non-specific binding sites with PBS solution containing 1% bovine serum albumin (BSA) at room temperature for 30 min, discard the blocking solution, add rabbit anti-CD44 primary antibody (concentration 1 µg / mL), and incubate at room temperature for 1 h; (3) Wash the cells three times with PBS, add FITC-labeled goat anti-rabbit IgG secondary antibody (concentration 1µg / mL), and incubate at room temperature in the dark for 1 h; finally, add DAPI staining solution to counterstain the cell nuclei, incubate in the dark, and then wash with PBS.

[0033] Observe and acquire images under a fluorescence microscope (see...) Figure 3 (a) In this study, the CD44 receptor was labeled with green fluorescence, and the cell nucleus was labeled with blue fluorescence. Compared with the PBS control group, the green fluorescence signal of the LPS-stimulated group was significantly enhanced, and the co-localization of green and blue cell nuclei was obvious in the Merge plot, indicating that LPS can effectively induce high expression of the CD44 receptor in RAW264.7 cells.

[0034] II. Verification of the Cell-Targeted Uptake Capacity of GLSO@HADA Nanoemulsion This experiment used near-infrared fluorescently labeled ursodeoxycholic acid-hyaluronic acid coupler (NIR@HADA) as the tracer material, where NIR is a near-infrared fluorescent dye and HADA is the ursodeoxycholic acid-hyaluronic acid coupler prepared in Example 1. The preparation method of NIR@HADA is the same as that of GLSO@HADA, except that Ganoderma lucidum spore oil is replaced with a near-infrared fluorescent dye, and its targeting core structure is exactly the same as that of GLSO@HADA. The targeting mechanism was verified by competitive inhibition experiments. Specific procedures are as follows: RAW264.7 cells were added at a concentration of 1×10⁻⁶ mL per well. 6 Cells were seeded at a density of [number] cells / mL in confocal culture dishes and incubated at 37°C for 24 h. Then, they were pretreated with 1 µg / mL LPS for 12 h to simulate an inflammatory microenvironment. Cells without LPS treatment were used as a background control. Cells were divided into three groups and treated as follows: (1) PBS control group: treated with PBS, and the subsequent treatment was the same as other groups; (2) LPS group: NIR@HADA (concentration 10mg / mL) was added directly and co-incubated with cells for 4h; (3) LPS+HA competitive inhibition group: First, add free hyaluronic acid (HA, concentration 10mg / mL) for pre-incubation for 2h to block the CD44 receptor on the cell surface, and then add NIR@HADA (concentration 10mg / mL) for co-incubation for 4h.

[0035] After incubation, the culture medium in each well was aspirated, and the cells were gently washed three times with pre-cooled PBS. Hoechst 33342 staining solution was added, and the cells were stained at room temperature in the dark for 15 minutes. The cells were washed three times again with PBS, and intracellular fluorescence images were observed and collected using a fluorescence microscope to analyze the uptake of nanoparticles.

[0036] Fluorescence image results as follows Figure 3 As shown in b, the blue fluorescence represents Hoechst 33342 cell nuclear staining, and the red fluorescence represents NIR@HADA. Cells in the PBS control group showed almost no red fluorescence, indicating minimal uptake of NIR@HADA under normal conditions. Strong red fluorescence was observed in the LPS group, indicating that cells can take up large amounts of NIR@HADA under inflammatory conditions. In contrast, the LPS+HA competitive inhibition group, pre-treated with free HA, showed a significant decrease in intracellular red fluorescence, confirming that NIR@HADA is primarily uptaken through the specific interaction between HA and the CD44 receptor. Combined with the electrostatic attraction between the negative charge on the nanoparticle surface and the inflammatory site, these results strongly suggest that GLSO@HADA nanoemulsion can precisely target activated macrophages at the site of intestinal inflammation, thereby improving the oxidative stress state of inflammatory cells by scavenging intracellular reactive oxygen species (ROS), providing reliable cellular-level evidence for in vivo targeted inflammation therapy using GLSO@HADA nanoemulsion.

[0037] Example 3 This embodiment uses a small animal in vivo imaging experiment to construct three fluorescently labeled tracer materials—NIR@GLSO, NIR@HA, and NIR@HADA—using Nile Red as the near-infrared fluorescent tracer dye. The in vivo targeting ability of the ursodeoxycholic acid-hyaluronic acid coupling compound (HADA) carrier system from Example 1 in a DSS-induced ulcerative colitis mouse model is evaluated. Specific experimental steps and results analysis are as follows: 1. Preparation of fluorescently labeled tracer materials Preparation of NIR@HADA and NIR@HA: Weigh 10 mg of Nile red and dissolve it in 50 mL of methanol to prepare Nile red methanol stock solution; weigh 30 mg of HADA and 30 mg of hyaluronic acid (HA) and dissolve them in 30 mL of deionized water to obtain carrier aqueous solutions; take 5 mL of Nile red methanol stock solution and add it to the above two carrier aqueous solutions, and stir at 12000 rpm for 10 min using a homogenizer, then stir with appropriate magnetic force at room temperature overnight to fully evaporate and remove residual methanol in the system, thus obtaining NIR@HADA and NIR@HA fluorescently labeled tracer materials.

[0038] Preparation of NIR@GLSO: Take 5 mL of the above Nile Red methanol stock solution and add it to 30 mL of Ganoderma lucidum spore oil (GLSO). Stir the mixture at 12000 rpm for 10 min using a homogenizer. Stir the mixture with appropriate magnetic force overnight at room temperature. Evaporate to remove residual methanol to obtain NIR@GLSO fluorescent labeling tracer material.

[0039] 2. Animal model construction and drug administration Twelve experimental mice were randomly divided into four groups of three mice each: control group; 3%DSS+NIR@GLSO group; 3%DSS+NIR@HA group; and 3%DSS+NIR@HADA group.

[0040] The control group was given free access to deionized water to establish a normal mouse model. The other three groups of mice were given free access to 3% sodium dextran sulfate (DSS) aqueous solution for 7 days to establish a mouse model of ulcerative colitis. On day 8 of modeling, the four groups of mice were administered oral medications via gavage. The control group was given physiological saline, while the other three groups were given equal doses of NIR@GLSO, NIR@HA, and NIR@HADA fluorescently labeled materials via gavage, respectively.

[0041] 3. In vivo imaging detection and result analysis Eight hours after drug administration, all mice were sacrificed and dissected. Heart, liver, spleen, lung, kidney, stomach, small intestine, and colon tissues were separated. Fluorescence distribution images of each tissue were acquired using a small animal in vivo imaging system, and the fluorescence intensity in the colon region was quantitatively analyzed. The results are as follows: Figure 4 As shown in the image, imaging results showed that no obvious fluorescence signals were observed in any tissue of the control group mice; in the 3% DSS model group, only weak fluorescence was observed in the colon region of the NIR@GLSO group and the NIR@HA group, and the fluorescence signal was diffusely distributed in normal intestinal tissues such as the stomach and small intestine; while the colon region of the NIR@HADA group mice showed a significantly stronger specific fluorescence signal than the other two groups, with fluorescence mainly concentrated in the inflamed area of ​​the colon, and no obvious non-specific fluorescence enrichment in other organs such as the heart, liver, spleen, lungs, and kidneys.

[0042] The above results fully demonstrate that the ursodeoxycholic acid-hyaluronic acid carrier system (HADA) of the present invention can achieve precise targeted enrichment of the colonic inflammation site in mice with ulcerative colitis by specifically binding HA to the CD44 receptor highly expressed in the colonic inflammation site.

[0043] Example 4 This embodiment uses the ABTS free radical scavenging experiment to systematically evaluate the in vitro antioxidant activity of the GLSO@HADA nanoemulsion prepared in Example 1.

[0044] ABTS (2,2'-adiazon-bis-3-ethylbenzothiazoline-6-sulfonic acid) is a stable synthetic free radical with a characteristic ultraviolet absorption peak at 734 nm, and the solution appears blue-green. When the free radical is scavenged by the sample, the absorbance at 734 nm will decrease accordingly. By detecting the change in absorbance, the scavenging rate of the sample on ABTS free radicals can be quantitatively calculated, thereby characterizing its antioxidant capacity.

[0045] The experimental procedure was as follows: ABTS was mixed with potassium persulfate to generate a stable blue-green ABTS radical stock solution. The stock solution was diluted with PBS buffer (pH 7.4) to an absorbance of 0.7-0.8 at 734 nm to obtain the ABTS radical working solution. GLSO@HADA nanoemulsion was added to the working solution to final concentrations of 4, 6, 8, 10, and 12 µg / mL, respectively. ABTS working solution with only an equal volume of PBS was used as a blank control. All samples were incubated in the dark for 10 min. After incubation, the supernatant was collected by centrifugation, and the absorbance at 734 nm was measured using a UV spectrophotometer to calculate the ABTS radical scavenging rate.

[0046] like Figure 5 As shown, the scavenging rate of ABTS free radicals by GLSO@HADA nanoemulsion exhibits a concentration-dependent increasing trend: at a concentration of 4 µg / mL, the scavenging rate is approximately 20%; at 6 µg / mL, the scavenging rate is approximately 38%; at 8 µg / mL, the scavenging rate is approximately 52%; at 10 µg / mL, the scavenging rate is approximately 70%; and at a concentration of 12 µg / mL, the scavenging rate can reach as high as approximately 83%. These results clearly demonstrate that the GLSO@HADA nanoemulsion of this invention possesses excellent free radical scavenging ability in vitro and can efficiently remove excess ABTS free radicals generated in the inflammatory microenvironment.

[0047] Example 5 This embodiment uses a cell model to evaluate the ability of the ursodeoxycholic acid-hyaluronic acid nanoemulsion (GLSO@HADA) prepared in Example 1 to scavenge reactive oxygen species (ROS) and protect cells from oxidative stress damage. Specific experiments and results are analyzed below: NCM460 cells (normal human colonic epithelial cells) were seeded in culture dishes and induced with 1 µg / mL lipopolysaccharide (LPS) and 1.5 mM hydrogen peroxide (H2O2) to construct a cell oxidative stress injury model. Subsequently, different samples were added for treatment, and the intracellular ROS level and cell viability were observed under a laser confocal microscope using the DCFH-DA ROS detection kit and the CalceinAM / PI cell liveness staining kit.

[0048] In ROS detection experiments ( Figure 6 a) Using DCFH-DA as a fluorescent probe, the intensity of green fluorescence directly reflects the intracellular ROS level. The control group showed no obvious green fluorescence, while the LPS-induced group treated with PBS exhibited significant green fluorescence, indicating that LPS can induce cells to produce a large amount of ROS. Compared with the HA, UDCA, HADA, and GLSO groups, the GLSO@HADA group showed a significantly weakened intracellular green fluorescence signal, with the lowest fluorescence intensity among all treatment groups. This suggests that GLSO@HADA can efficiently clear LPS-induced intracellular ROS, significantly superior to single-component and control vectors.

[0049] In cell viability staining experiments ( Figure 6 (b) Live cells were labeled with green fluorescence (Calcein AM), and dead cells were labeled with red fluorescence (PI). Control group cells showed intact green fluorescence, while the H2O2-induced group treated with PBS showed abundant red fluorescence, indicating that H2O2 caused severe cell death. Comparing the treatment groups, the GLSO@HADA group showed almost no red fluorescence, with green fluorescence signals similar to the control group, and intact cell morphology; while the HA, UDCA, HADA, and GLSO groups all showed varying degrees of red dead cell signals.

[0050] The above results fully demonstrate that the GLSO@HADA nanoemulsion of the present invention can significantly reduce LPS and H2O2-induced oxidative stress damage by efficiently removing excess ROS in cells, while effectively protecting the activity of colonic epithelial cells and reducing cell death rate, providing solid cell experimental evidence for its ability to alleviate colitis and repair intestinal mucosal damage in vivo.

[0051] Example 6 This embodiment systematically evaluates the in vivo preventive anti-inflammatory and tissue protective effects of the ursodeoxycholic acid-hyaluronic acid nanoemulsion (GLSO@HADA) prepared in Example 1 using a DSS-induced mouse ulcerative colitis model. The specific experimental steps and results analysis are as follows: Female C57BL / 6J mice aged 5-6 weeks were randomly divided into 8 groups after acclimatization for 1 week. The grouping and treatment were as follows: (1) Control group: PBS was administered by gavage + normal drinking water; (2) DSS group: PBS was administered by gavage + 3% sodium dextran sulfate (DSS) in drinking water; (3) 5-ASA group: 5-aminosalicylic acid (clinical drug, positive control, 40 mg / kg) was administered by gavage + 3% DSS in drinking water; (4) HA group: hyaluronic acid (HA, 40 mg / kg) was administered by gavage + 3% DSS in drinking water; (5) UDC group: 5-aminosalicylic acid (HA, 40 mg / kg) was administered by gavage + 3% DSS in drinking water; Group A: Ursodeoxycholic acid (UDCA, 40 mg / kg) was administered by gavage + 3% DSS in drinking water; (6) HADA group: Ursodeoxycholic acid-hyaluronic acid coupling compound (HADA, 40 mg / kg) was administered by gavage + 3% DSS in drinking water; (7) GLSO group: Ganoderma lucidum spore oil (GLSO, 40 mg / kg) was administered by gavage + 3% DSS in drinking water; (8) GLSO@HADA group: Ursodeoxycholic acid-hyaluronic acid nanoemulsion loaded with Ganoderma lucidum spore oil (GLSO@HADA, 40 mg / kg) was administered by gavage + 3% DSS in drinking water.

[0052] Except for the Control group, mice in all other groups were given 3% DSS aqueous solution for 7 consecutive days to establish an ulcerative colitis model. The drug treatment groups were given oral gavage on the first day of model establishment (day 1) (days 1, 3, 5, and 7) to prevent the development of colitis. The Control group and the DSS group were given the same amount of PBS by gavage. The weight changes of mice were recorded daily during the experiment. On day 9, all mice were euthanized, and the colon tissue was dissected, and the colon length was measured and counted.

[0053] like Figure 7 As shown, GLSO@HADA nanoemulsion can significantly prevent and alleviate DSS-induced colitis symptoms: Figure 7 (a) shows the weight change curve of mice. The weight of mice in the DSS group continued to decrease with the increase of the modeling days, while the weight of mice in the GLSO@HADA group stopped decreasing from day 5 and gradually increased in subsequent experimental cycles. The weight change trend of the GLSO@HADA group was significantly better than that of the DSS group, the single component (HA, UDCA, GLSO) and the HADA group, and was close to the level of the healthy control group. This indicates that GLSO@HADA can effectively prevent the progression of colitis and improve the overall health status of mice. Figure 7 (b) and Figure 7(c) shows the statistics and representative photos of mouse colon length. DSS treatment led to a significant shortening of the mouse colon, indicating severe intestinal inflammation and tissue damage. The colon length of the GLSO@HADA group was not significantly different from that of the Control group, but was significantly longer than that of the DSS group and other treatment groups. It was comparable to or better than the positive drug 5-ASA group, indicating that GLSO@HADA can effectively prevent colon tissue damage and reduce inflammation-mediated colon shortening.

[0054] The above results fully demonstrate that the GLSO@HADA nanoemulsion of the present invention can significantly alleviate typical colitis symptoms such as weight loss and colon shortening in a DSS-induced ulcerative colitis mouse model through early preventive intervention, targeting the site of colonic inflammation, clearing reactive oxygen species, and regulating the inflammatory microenvironment. It has excellent in vivo preventive anti-inflammatory and tissue protective effects, providing reliable animal experimental evidence for its application in the prevention and clinical treatment of ulcerative colitis.

[0055] Example 7 This embodiment systematically evaluates the delayed therapeutic effect of the ursodeoxycholic acid-hyaluronic acid nanoemulsion (GLSO@HADA) prepared in Example 1 on pre-existing colitis using a DSS-induced mouse ulcerative colitis (UC) model. Specific experiments and results are analyzed below: Female C57BL / 6J mice aged 5-6 weeks were randomly divided into eight groups after acclimatization for one week. The grouping and treatment were as follows: (1) Control group: PBS gavage + normal drinking water; (2) DSS group: PBS gavage + 3% sodium dextran sulfate (DSS) drinking water; (3) 5-ASA group: 5-aminosalicylic acid (positive control, 40 mg / kg) gavage + 3% DSS drinking water; (4) HA group: hyaluronic acid (40 mg / kg) gavage + 3% DSS. Drinking water; (5) UDCA group: Ursodeoxycholic acid (40mg / kg) by gavage + 3% DSS in drinking water; (6) HADA group: Ursodeoxycholic acid-hyaluronic acid (40mg / kg) by gavage + 3% DSS in drinking water; (7) GLSO group: Ganoderma lucidum spore oil (40mg / kg) by gavage + 3% DSS in drinking water; (8) GLSO@HADA group: Ursodeoxycholic acid-hyaluronic acid nanoemulsion loaded with Ganoderma lucidum spore oil (40mg / kg) by gavage + 3% DSS in drinking water.

[0056] All experimental mice were given a 3% DSS aqueous solution to establish an ulcerative colitis model from day 0 to day 7, after which they were given normal drinking water. The drug treatment groups were administered the drug by gavage on days 7, 9, 13, and 15, while the control group and the DSS group were administered the same volume of PBS by gavage. The weight changes of the mice were recorded daily during the experiment. On day 15, the mice were euthanized, and the colon and related tissues were dissected to detect colon length and serum biochemical indicators.

[0057] like Figure 8 As shown, GLSO@HADA nanoemulsion can significantly alleviate DSS-induced colitis symptoms and repair tissue damage: Figure 8 (a) shows the weight change curve of mice. The weight of mice in the DSS model group continued to decrease as the modeling progressed, while each treatment group could alleviate the weight loss to varying degrees. Among them, the weight loss of mice in the GLSO@HADA group was the smallest and the recovery speed was the fastest. From day 7 (end of modeling and start of drug administration), the weight stopped decreasing and gradually recovered. The overall condition was significantly better than other treatment groups and close to the level of the healthy control group, indicating that GLSO@HADA can effectively improve the overall health status of colitis mice. Figure 8 (b) and Figure 8 (c) shows the statistics of colon length in mice and representative anatomical photographs. The colon in the DSS group was significantly shortened due to inflammatory damage, while the colon length in the GLSO@HADA group was not significantly different from that in the Control group, but was significantly longer than that in the DSS group and other single-component treatment groups. The efficacy was comparable to or better than that in the positive drug 5-ASA group, which confirms that GLSO@HADA can effectively repair colonic inflammatory damage and restore colon length.

[0058] The above results fully demonstrate that the GLSO@HADA nanoemulsion of the present invention has excellent delayed treatment effect on existing colitis in a DSS-induced ulcerative colitis mouse model, and can effectively alleviate weight loss and restore colon length.

[0059] Example 8 Based on the DSS-induced delayed treatment model of ulcerative colitis in mice in Example 7, this embodiment further evaluates the protective effect of the ursodeoxycholic acid-hyaluronic acid nanoemulsion (GLSO@HADA) prepared in Example 1 on intestinal barrier function and liver function.

[0060] The grouping, modeling, and drug administration of experimental animals were the same as in Example 7.

[0061] On day 14 of the experiment, intestinal permeability was tested in a subset of mice: 4 kDa FITC-glucan (2 mg / kg) was administered orally by gavage. Two hours later, blood was collected from the orbital cavity, and plasma was separated by centrifugation at 3000g for 10 minutes. Plasma fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The amount of FITC-glucan leakage reflected the integrity of the intestinal barrier. Blood was collected from the orbital cavity of the remaining mice on day 15. Serum was separated, and lipopolysaccharide (LPS), total bile acids (TBAs), triglycerides (TG), total cholesterol (TC), and liver function-related biochemical indicators (AST, ALP, LDH, ALT) were measured.

[0062] like Figure 9 As shown, GLSO@HADA nanoemulsion can significantly improve intestinal barrier function and protect liver function: Figure 9 (a) shows the results of FITC-glucan fluorescence intensity detection. The fluorescence intensity of FITC-glucan in plasma of the DSS group was significantly increased, indicating increased intestinal permeability and impaired barrier function. In contrast, the fluorescence intensity of the GLSO@HADA group was significantly reduced, close to the level of the Control group, indicating that GLSO@HADA can effectively repair the intestinal barrier and reduce FITC-glucan leakage. Figure 9 (be) represents the results of serum inflammation and metabolism-related indicators. In the DSS group, the levels of serum LPS, total bile acids (TBAs), triglycerides (TG), and total cholesterol (TC) were significantly elevated, indicating bacterial endotoxin leakage and metabolic disorders. In contrast, the GLSO@HADA group showed significant reductions in the above indicators, approaching the levels of the Control group, confirming that GLSO@HADA can effectively inhibit bacterial translocation and reduce endotoxin leakage and metabolic abnormalities. Figure 9 (f) shows the results of liver function-related biochemical indicators. The DSS group showed significantly elevated levels of AST, ALP, LDH, and ALT, indicating liver function damage. However, the GLSO@HADA group showed no significant difference in the above liver function indicators compared to the Control group, indicating that GLSO@HADA exerts its anti-inflammatory therapeutic effect without causing additional liver burden and has good biosafety.

[0063] The above results fully demonstrate that the GLSO@HADA nanoemulsion of the present invention can significantly reduce DSS-induced gut-liver axis damage and improve liver function indicators by repairing intestinal barrier integrity, inhibiting bacterial translocation and endotoxin leakage.

Claims

1. A method for preparing an ursodeoxycholic acid-hyaluronic acid nanoemulsion, characterized in that: First, hyaluronic acid and ursodeoxycholic acid are covalently linked through a two-step amidation reaction to obtain an ursodeoxycholic acid-hyaluronic acid coupling compound. Then, the coupling compound is used as a carrier to emulsify with a fat-soluble antioxidant oil to obtain an ursodeoxycholic acid-hyaluronic acid nanoemulsion.

2. The method for preparing ursodeoxycholic acid-hyaluronic acid nanoemulsion according to claim 1, characterized in that, Specifically, the steps include the following: 1) Hyaluronic acid was dissolved in deionized water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added and stirred to activate the solution. Then, ethylenediamine was added to carry out the first-step amidation reaction. The reaction solution was dialyzed and lyophilized to obtain hyaluronic acid-ethylenediamine. 2) Hyaluronic acid-ethylenediamine was dissolved in deionized water to obtain solution 1; ursodeoxycholic acid was dissolved in N,N-dimethylformamide, and EDC and NHS were added and stirred to activate the solution to obtain solution 2; solution 1 was slowly added to solution 2 to carry out the second amidation reaction, and the reaction solution was dialyzed and lyophilized to obtain ursodeoxycholic acid-hyaluronic acid coupling compound; 3) Dissolve the ursodeoxycholic acid-hyaluronic acid coupling compound in deionized water, add lipid-soluble antioxidant oil, and stir using a homogenizer to obtain ursodeoxycholic acid-hyaluronic acid nanoemulsion.

3. The method for preparing ursodeoxycholic acid-hyaluronic acid nanoemulsion according to claim 1 or 2, characterized in that: The fat-soluble antioxidant oil includes one or more of the following: Ganoderma lucidum spore oil, vitamin E, rice bran oil, and fish oil.

4. The method for preparing ursodeoxycholic acid-hyaluronic acid nanoemulsion according to claim 2, characterized in that: In step 1), the molar ratio of hyaluronic acid, ethylenediamine, NHS, and EDC is 1-2:1-3:1-2:1-2.

5. The method for preparing ursodeoxycholic acid-hyaluronic acid nanoemulsion according to claim 2, characterized in that: In step 2), the molar ratio of ursodeoxycholic acid, hyaluronic acid-ethylenediamine, NHS and EDC is 1-2:1-3:1-2:1-2.

6. The method for preparing ursodeoxycholic acid-hyaluronic acid nanoemulsion according to claim 1, characterized in that: In steps 1) and 2), the molecular weight cutoff of the dialysis bag used for dialysis is 10 kDA-20 kDA.

7. The method for preparing ursodeoxycholic acid-hyaluronic acid nanoemulsion according to claim 1, characterized in that: In step 3), the ratio of ursodeoxycholic acid-hyaluronic acid coupling compound, deionized water and fat-soluble antioxidant oil is 20-80 mg: 20-100 mL: 5-20 mL.

8. The method for preparing ursodeoxycholic acid-hyaluronic acid nanoemulsion according to claim 1, characterized in that: In step 3), the homogenizer rotates at 10,000 to 12,000 rpm and the mixing time is 15 to 40 minutes.

9. An ursodeoxycholic acid-hyaluronic acid nanoemulsion, characterized in that, It is prepared by the method described in any one of claims 1 to 8.

10. The application of the ursodeoxycholic acid-hyaluronic acid nanoemulsion according to claim 9, characterized in that: Used to prepare drugs for the prevention and / or treatment of inflammatory bowel disease.

Images