Nano co-delivery drug delivery carrier loaded with triptolide and application of nano co-delivery drug delivery carrier in preparation of medicine for treating interstitial cystitis

Abstract

The invention belongs to the technical field of exosome application, and discloses a triptolide-loaded nano co-delivery drug delivery carrier and application thereof in preparation of drugs for treating interstitial cystitis. The triptolide-loaded nano co-delivery drug delivery carrier is used for interstitial cystitis model animals, and the following changes are observed: 1, the sensitivity of the model animals to stimulation is reduced; 2, the urination frequency of model animals is reduced, and the urination volume of words is increased; 3, the number of red blood cells in urine of the model animal is reduced; 4, bladder tissue hyperemia is relieved, and the size is reduced; and 5, the expression of inflammatory factors IL-6, MCP-1, ICAM-1 and BAX is obviously reduced. Therefore, it can be determined that the triptolide-loaded nano-co-delivery drug delivery carrier is used for treating interstitial cystitis. According to the research, the application range of the triptolide is expanded, and the triptolide is combined with the exosome to be used for treating the interstitial cystitis, so that a new thought is provided for treating the interstitial cystitis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of exosome application, and more particularly relates to a nano co-delivery drug carrier loaded with triptolide and application thereof in preparation of a drug for treating interstitial cystitis. BACKGROUND

[0002] Interstitial cystitis (IC) is a bladder pain disease related to urination function, with unknown causes, accompanied by irritating symptoms such as frequent urination, urinary urgency and bladder or pelvic pain. IC can occur in people of all ages, and women are more common. In recent years, the diagnosis rate of the disease in China has increased year by year, and the quality of life of IC patients has been seriously affected.

[0003] Exosomes are nanoscale (30-150 nm) extracellular vesicles secreted by cells, which were once thought to be a way for cells to excrete waste. But now the scientific community realizes that exosomes are key messengers of intercellular communication, carrying important functional molecules such as proteins, lipids, RNA (such as mRNA, miRNA) and DNA, and delivering them from the source cell to the recipient cell, thereby changing the function of the recipient cell. Based on this natural mechanism, exosomes have shown great potential in disease treatment, and their therapeutic effects mainly manifest in the following aspects: 1. Natural messenger and functional regulator: The biological active substances (such as anti-inflammatory miRNA, pro-angiogenic factors, growth factors, etc.) carried by exosomes can directly regulate the physiological and pathological processes of recipient cells, such as inhibiting inflammation, promoting tissue repair, regulating immune response, etc. 2. Excellent drug delivery carrier: Exosomes have natural nanoscale size and good biocompatibility, and can cross physiological barriers such as the blood-brain barrier. They can be engineered to load specific therapeutic drugs (such as small molecule drugs, nucleic acid drugs siRNA / miRNA, mRNA, etc.), and use their surface targeting proteins to precisely deliver drugs to diseased cells (such as cancer cells, damaged neurons), achieving efficient and low-toxicity targeted therapy.

[0004] Triptolide (TPL) is an epoxy diterpene lactone compound isolated from the roots and rhizomes of the Chinese herb Triptygium wilfordii Hook F. It has anti-inflammatory, analgesic, immunomodulatory, and anti-tumor effects. However, TPL has poor solubility, a narrow therapeutic window, and strong hepatotoxicity and nephrotoxicity, which severely limit its clinical application. To safely apply the strong biological activity of TPL in clinical practice, the existing technology often modifies the molecular structure of TPL through chemical methods to reduce its toxicity while retaining or enhancing its efficacy, resulting in compounds such as Celastrol and Minnelide (a prodrug), among others. Minnelide has been used in clinical trials for the treatment of pancreatic cancer and other malignant tumors.

[0005] Exosomes are a new type of drug delivery system that has been discussed more in recent years. The core technology is to encapsulate drugs in exosomes to achieve targeted delivery. Using exosomes as drug delivery carriers has many benefits, such as improving treatment targeting: reducing damage to normal tissues; reducing systemic toxicity: enhancing drug bioavailability, allowing lower doses to be used; overcoming drug resistance: nanocarriers can help bypass certain drug resistance mechanisms of tumors.

[0006] The existing technology lacks a technical solution for a drug delivery system formed by loading TPL into umbilical cord mesenchymal stem cell exosomes for treating interstitial cystitis. SUMMARY

[0007] Based on the above-mentioned defects existing in the prior art, the present application first provides a nano co-delivery drug carrier loaded with TPL.

[0008] A second object of the present application is to provide the use of the above-mentioned nano co-delivery drug carrier loaded with TPL.

[0009] The object of the present application is achieved by the following technical solutions:

[0010] The inventive concept of the present application is that in the preliminary exploration experiment, the inventors fed a small amount of TPL to model animals with interstitial cystitis and found that it could relieve the urination disorders of the model animals, specifically manifested by a decrease in the number of urinations and an increase in the amount of single urination. Given the high toxicity of TPL, in order to improve its application effect on interstitial cystitis, the inventive concept mixes TPL with umbilical cord mesenchymal stem cell exosomes to obtain TPL loaded umbilical cord mesenchymal stem cell exosomes, which has a significant application effect on interstitial cystitis and has promotional application value.

[0011] Therefore, the present application first provides a triptolide-loaded nanocarrier for co-delivery, which is prepared by mixing equal amounts of umbilical cord mesenchymal stem cell exosomes and triptolide.

[0012] Preferably, the particle size of the triptolide-loaded nanocarrier for co-delivery is 72-91 nm.

[0013] Preferably, the amount of triptolide is 25 ng / mL.

[0014] Preferably, the triptolide-loaded nanocarrier for co-delivery of the present application can be prepared by co-incubation, ultrasonication, electroporation, freeze-thaw cycle, chemical transfection reagent, etc.

[0015] Considering that the ultrasonic method and the electroporation method may cause a certain degree of damage to the exosome membrane, more preferably, the present application uses the co-incubation method to load TPL.

[0016] As a specific embodiment, the preparation method of the triptolide-loaded nanocarrier for co-delivery of the present application is as follows: after the exosome is purified and identified, it is resuspended with PBS, and then an equal amount of TPL (TPL concentration is 25 ng / mL) is added, and the incubation is carried out on a shaker for 100 min at a speed of 200 rpm. The free and non-specifically bound triptolide is removed by ultracentrifugation filtration, and the triptolide-loaded nanocarrier for co-delivery is obtained.

[0017] The triptolide-loaded nanocarrier for co-delivery prepared by the present application is used for interstitial cystitis model animals, and the following changes are observed: 1. The sensitivity of the model animals to stimulation is reduced; 2. The number of urination of the model animals is reduced, and the single-word urination volume is increased; 3. The number of red blood cells in the urine of the model animals is reduced; 4. The congestion of the bladder tissue is relieved, and the volume is reduced; 5. The expression of inflammatory factors IL-6, MCP-1, ICAM-1 and BAX is significantly reduced. Therefore, it can be determined that the triptolide-loaded nanocarrier for co-delivery is used for treating interstitial cystitis.

[0018] Therefore, the present application also provides the use of the triptolide-loaded nanocarrier for co-delivery in the preparation of a drug for treating and / or relieving interstitial cystitis.

[0019] The present application also provides the use of the triptolide-loaded nanocarrier for co-delivery in the preparation of a drug for relieving and / or inhibiting inflammation of interstitial cystitis.

[0020] Preferably, the triptolide-loaded nanocarrier for co-delivery inhibits the expression of IL-6, MCP-1, ICAM-1 and BAX in inflammation.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The application provides a triptolide-loaded nanocarrier for co-delivery, which is used for an interstitial cystitis model animal, and the following changes are observed: 1, the model animal is less sensitive to stimulation; 2, the model animal urinates less frequently and increases the single-word urination volume; 3, the number of red blood cells in the urine of the model animal is reduced; 4, the congestion of the bladder tissue is relieved and the volume is reduced; 5, the expression of inflammatory factors IL-6, MCP-1, ICAM-1 and BAX is significantly reduced. Therefore, it can be determined that the triptolide-loaded nanocarrier for co-delivery is used for treating interstitial cystitis. The present research expands the application range of triptolide, and combines the exosome with triptolide to treat interstitial cystitis, thereby providing a new idea for the treatment of interstitial cystitis. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The figure is a particle size distribution diagram of umbilical cord mesenchymal stem cell exosomes;

[0024] Figure 2 The figure is an electron microscope diagram of TPL-exo nanoparticles (the scale is 80 μm);

[0025] Figure 3 The figure is an animal Von Frey experiment diagram;

[0026] Figure 4 The figure is an animal urine behavior performance diagram;

[0027] Figure 5 The figure is an animal urine red blood cell content diagram (+ indicates detection, and - indicates negative and no detection);

[0028] Figure 6 The figure is an animal bladder shape;

[0029] Figure 7 The figure is an animal bladder tissue HE staining diagram (the upper diagram is enlarged by 2X, and the field of view is 1500 um; the corresponding lower diagram is enlarged by 20X, and the field of view is 150 um);

[0030] Figure 8 The figure is a statistical diagram of the expression amounts of animal bladder tissue cytokines IL-6, MCP-1, ICAM-1 and BAX. DETAILED DESCRIPTION

[0031] In order to better illustrate the purposes, technical solutions and advantages of the application, the application will be further described below in combination with specific drawings and examples. In the examples, the experimental methods used are conventional methods, and the materials, reagents and the like used are commercially available, unless otherwise specified.

[0032] Example 1 Preparation of triptolide-loaded umbilical cord mesenchymal stem cell exosomes (TPL-exo)

[0033] I. Preparation of umbilical cord mesenchymal stem cell exosomes

[0034] Exosomes were extracted from umbilical cord mesenchymal stem cell supernatant using ultracentrifugation. After extraction, the concentration and vesicle purity of the exosomes were detected using a nanoflow instrument.

[0035] Extraction method: Take 20 ml of umbilical cord mesenchymal stem cell supernatant, centrifuge at 10,000 g for 30 minutes, take the supernatant, centrifuge at 100,000 g for 90 minutes, discard the precipitate, resuspend the supernatant with 1 ml of 1x PBS and mix well, and the exosomes are obtained.

[0036] Nanoflow instrument detection conditions: Take 50 μl of exosome sample, detect on the machine, record the particle number within 60 s, save the report, and the software calculates the particle size and concentration of the exosome sample according to the data of the concentration standard and the particle standard.

[0037] The results are shown in Figure 1 The particle size range of the exosome sample is shown in

[0038] II. Preparation of triptolide-loaded umbilical cord mesenchymal stem cell exosomes (TPL-exo)

[0039] After purification and identification of the exosomes, resuspend them in PBS, add an equal amount of TPL (TPL concentrations are 5 ng / mL, 15 ng / mL, 25 ng / mL and 30 ng / mL), and incubate on a shaker at 200 rpm for 100 min. Remove the free and non-specifically bound triptolide by ultracentrifugation filtration.

[0040] The TEM image of the obtained TPL-exo is shown in Figure 2 It can be seen that the average particle size of TPL-exo is 85 nm.

[0041] 1. Drug loading, encapsulation efficiency and in vitro release of TPL-exo

[0042] Encapsulation efficiency (EE) and drug loading (DL) are important indicators for evaluating drug preparations. Encapsulation efficiency is the percentage of the amount of drug encapsulated in the carrier relative to the total amount of drug input. The formula is EE (%) = (Wt-Wf) / Wt x 100, where Wt and Wf are the total drug mass and the mass of free drug, respectively.

[0043] Drug loading is the mass of drug loaded in the carrier divided by the mass of the carrier and the mass of drug loaded, formula is DL (%) = We / Wm x 100%, We represents the mass of drug encapsulated in the carrier; Wm represents the total mass of drug-loaded carrier.

[0044] The release behavior of TPL in TPL-exo was determined by dialysis method. The dialysis bag (MWCO 10 kDa) was cut into 6 cm long pieces, boiled in distilled water for 20 min, then washed with distilled water, stored at 4°C, and ensured that the dialysis bag was always immersed in the solution. 2 mL TPL-exo solution was transferred into the dialysis bag, the two ends were tightly tied, and the dialysis bag was placed in 50 mL PBS containing 0.1% (w / v) Tween 80 at pH 5.5 and pH 7.2, and placed in a constant temperature water bath shaker (37°C, 100 r / min). 5 mL samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12 and 24, respectively, for testing, while the same volume of fresh release medium at the same temperature was added, the sample medium was concentrated, and the cumulative release rate of TPL was calculated by HPLC.

[0045] HPLC was used to determine the drug loading and encapsulation efficiency of exo-TPL nanoparticles. The in vitro drug release performance of exo-TPL nanoparticles was determined by HPLC method, and the results are shown in Table 1.

[0046] Table 1 Drug loading and encapsulation efficiency of exo-TPL nanoparticles

[0047] Regarding the encapsulation efficiency and drug loading, from Table 1, it can be seen that the encapsulation efficiency shows a downward trend, which may be due to the increase in drug concentration exceeding the loading range of exosomes, or the high concentration of drug reducing the fluidity of the exosome membrane. More drugs may be attached to the exosome membrane, resulting in an increase in drug loading. The drug loading and encapsulation efficiency of TPL-exo nanoparticles prepared by mixing 15 ng / mL, 25 ng / mL and 30 ng / mL TPL with exosomes are higher, and the in vitro drug release performance is good; among them, the TPL-exo nanoparticles prepared by 25 ng / mL TPL have the best comprehensive performance.

[0048] The current mainstream theory and a large amount of research evidence show that the bladder wall tissue of interstitial cystitis, especially the interstitial region, is in a relatively acidic microenvironment, therefore, the present application also investigates the influence of TPL-exo nanoparticles on drug release in an acidic environment and a normal physiological environment, and Table 1 shows that the release rate of TPL-exo nanoparticles in the PBS dialysate with pH 7.2 is lower than that in the PBS dialysate with pH 5.5, which indicates that the TPL-exo nanoparticles prepared in the present application are more suitable for an acidic microenvironment and can ensure a higher drug release rate. Moreover, the TPL-exo nanoparticles prepared from 25 ng / mL TPL have the optimal release performance.

[0049] 2. MTT method for determining cytotoxicity

[0050] Culture and subculture of uroepithelial cells: a complex culture medium was prepared by adding 10% fetal bovine serum, streptomycin (100 μg / ml) and penicillin (100 U / mL) to F12K medium, and then uroepithelial cells (ATCC; CRL-9520) were quickly recovered at 37°C and transferred to the prepared F12K medium, and then incubated in a 37°C incubator with 95% air / 5% CO2. The cell state and density were observed daily, and when the cell growth area was 50%-60% of the bottom area of a 10 cm culture dish, the medium was replaced, and when the cell density was more than 90%, the cells were subcultured at a ratio of 1:2, and the subcultured cells were labeled with time and corresponding generation number.

[0051] The uroepithelial cells were inoculated in a 96-well plate at 100 μL per well, and the cell number was about 1×10 4 , and then routinely cultured for 24 h. Except that the control group was added with normal saline, the other three groups were respectively given TPL-exo nanoparticles, TPL and blank nanoparticles exosme for continuous culture for 24 h and 48 h. The supernatant was discarded, the cells were washed with PBS, and then MTT 20 μL and serum-free medium 80 μL were added, and then incubated at 37°C for 4 h. The supernatant was centrifuged and discarded, DMSO 150 μL was added to each well, and then placed on a shaker for low-speed shaking for 10 min, and then the absorbance value at 570 nm was detected by an enzyme-labeled instrument. The cell survival rate was calculated according to the formula. The formula is as follows: living cell rate (%) = (absorbance value of experimental group / absorbance value of control group) x 100%.

[0052] From the results of Table 2, it can be seen that as the concentration of TPL increases, the inhibition rate of cells is more and more obvious, and the toxicity of 30 ng / mL TPL to cells is more obvious. When TPL-exo nanoparticles are prepared by mixing exosome and TPL, the toxicity of TPL-exo nanoparticles to cells is significantly reduced. However, the toxicity of TPL-exo nanoparticles prepared by 30 ng / mL TPL to cells is significantly enhanced compared with TPL-exo nanoparticles prepared by 15 ng / mL TPL and TPL-exo nanoparticles prepared by 25 ng / mL TPL. It is possible that the drug loading of TPL-exo nanoparticles prepared by 30 ng / mL TPL is high, but the encapsulation efficiency does not improve, resulting in a small part of TPL exposed outside the exosome, thereby having a significant adverse effect on cell survival rate.

[0053] Table 2 Comparison of cell survival rate

[0054] According to the data in Table 1 and Table 2, TPL-exo nanoparticles prepared by 25 ng / mL TPL are selected for the following experiments to investigate the therapeutic effect on interstitial cystitis.

[0055] Example 2 Animal experiment

[0056] The CYP was used to construct an animal model, and the specific operation was as follows: 7-week-old SD female rats with a body weight of 250-350 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were kept in a condition of 21±3℃, and experienced 12h light / dark cycle, free feeding and drinking water, and at least 3d adaptation to the experimental environment.

[0057] A total of 28 experimental rats were divided into 4 groups, blank group: intraperitoneal injection of 0.9% NaCl; model group: intraperitoneal injection of 125mg / kg CYP; exo group: intraperitoneal injection of 125mg / kg CYP+ tail vein injection of 25 ng / mL exosome once a day; TPL-exo group: intraperitoneal injection of 125mg / kg CYP+ tail vein injection of 25 ng / mL TPL-exo once a day, and the behavior of the animals was observed every day, and the animals were treated after 4d, and dissected.

[0058] I. Fibrous filament allodynia experiment

[0059] Mechanical stimulation of the abdomen was performed in rats using 8 von Frey fiber filaments of different stiffness to assess the pain response. Before testing, the designated abdominal area of each rat was shaved, and the animals were placed in a transparent Plexiglas box on an elevated mesh floor and allowed to acclimate for at least 30 min. During the von Frey test, each fiber was applied through the mesh with a force that was just able to bend it slightly for 1-2 s, this procedure was repeated 3 times for each fiber with 5 s intervals, and the stimulation area close to the bladder was carefully changed to avoid desensitization. The pain response was scored as follows: 0 = no response; 1 = abdominal constriction; 2 = constriction plus change in position; 3 = constriction, change in position, licking of the stimulation area and / or vocalization. The pain score was calculated based on the maximum pain response score exhibited by the animal in the three stimulations.

[0060] II. Urine spot test

[0061] During the experiment, SD rats were placed in a standard cage for 4 h, the bedding was replaced with Whatman No. 3 filter paper, and the rats could freely eat and drink, then the urine traces were photographed under ultraviolet light, and the percentage of the area covered by the urine traces was recorded, and the area of the urine traces was analyzed and calculated using Image J software.

[0062] The results are shown in Figure 3 , and compared with the blank group, the model group animals were more sensitive to stimulation, while the exo group could alleviate the sensitivity to a certain extent, but the animals in the TPL-exo group had a great relief in the stimulation sensitivity, even close to the blank group, thus it can be seen that the pain response of the animals in the TPL-exo group was greatly reduced, and compared with the exo group, the TPL-exo group could significantly treat the inflammatory response of the model animals.

[0063] Looking at the Figure 4 , Figure 4 The left side shows the average number of urination of animals in each group, and the right side shows the area of urine spot on paper of animals in each group; the left side shows that compared with the blank group, the average number of urination of animals in the model group increased significantly, while the average number of urination of animals in the treatment group (exo group and TPL-exo group) was alleviated, and compared with the exo group, the average number of urination of animals in the TPL-exo group was greatly reduced; the right side shows that compared with the blank group, the urine volume of animals in the model group was significantly reduced, while the urine volume of animals in the treatment group (exo group and TPL-exo group) was significantly increased, and compared with the exo group, the urine volume of animals in the TPL-exo group increased more significantly, closest to the blank group. From the urination behavior of Figure 4 , it can be seen that the TPL-exo group can effectively alleviate the cystitis performance of the model animals.

[0064] Figure 5The results of collecting urine of each group of animals and detecting the amount of red blood cells in the urine composition are shown in Table 1. Figure 5 As can be seen from Table 1, compared with the blank group, the model group animals have a large amount of red blood cells in the urine, which indicates that the model animals have obvious urinary tract infection, leading to mucosal hyperemia and hemorrhage; compared with the model group, the amount of red blood cells in the urine of the treatment group (exo group and TPL-exo group) animals is greatly reduced, among which, the urine of two animals in the exo group still contains a small amount of red blood cells, indicating that the inflammation of the exo group animals is partially alleviated, and no obvious red blood cells are detected in the urine of the TPL-exo group animals, indicating that TPL-exo can effectively treat urinary tract infection of the model animals.

[0065] Based on the above experimental observations, it can be determined that the treatment effect of TPL-exo treated animals on cystitis is more significant than that of exo treated animals. In the following, we will focus on the pathological manifestations and expression changes of inflammatory factors of TPL-exo treated animals to lay a foundation for studying the mechanism of treatment of interstitial cystitis.

[0066] III. HE Staining

[0067] After 4 days of observation, the rats were dissected, and the bladder tissues of the animals in the blank group, the model group and the TPL-exo group were photographed and recorded, and the following treatments were performed: 20 min in dimethylbenzene I, 20 min in dimethylbenzene II, 5 min in anhydrous ethanol I, 5 min in anhydrous ethanol II, 5 min in 75% alcohol, and 5 min in distilled water. The slices were placed in hematoxylin staining solution for 3-8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, washed with tap water, then returned to blue with 0.6% ammonia water, and washed with running water. Then the slices were sequentially placed in 85% and 95% gradient alcohol for 5 min each, stained with eosin for 1-3 min. Then the slices were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, dimethylbenzene I for 5 min, dimethylbenzene II for 5 min, and sealed with neutral resin, and observed under an optical microscope.

[0068] Figure 6 The appearance and size of the removed bladders of the rats in each group are shown in Table 2. Figure 6 As can be seen from Table 2, compared with the blank group, the bladders of the rats in the model group have obvious hyperemia, the volume of the bladder is obviously larger, and the mucosa is thinner and more transparent; compared with the model group, the bladder tissue of the TPL-exo group tends to be smaller, and the hyperemia is greatly alleviated, and even the size of the bladder of some rats is similar to that of the blank group, and the dissection diagram also verifies that TPL-exo treatment indeed has a significant therapeutic effect on cystitis.

[0069] HE staining of bladder tissue as Figure 7 As shown in FIG. 6, the model group showed more inflammatory cell infiltration in the bladder mucosa, and the mucosa was obviously edematous, and there were more degranulation mast cells, which was consistent with the typical IC pathological characteristics. The bladder mucosa of the blank group had a small amount of inflammatory cell infiltration, and the mucosa and stroma were not edematous or dilated and congested with capillaries, and no degranulation mast cells were observed. The mucosa of the TPL-exo group showed no obvious damage, and the stroma was dilated and congested with capillaries, and a small amount of inflammatory cells infiltrated. It can also be clearly seen from the histopathological graph that TPL-exo treatment can significantly alleviate the mucosal edema, inflammatory cell infiltration, and mast cell degranulation caused by cystitis.

[0070] IV. Detection of the expression amount of cytokines in bladder tissue

[0071] ICAM-1 determination in bladder tissue: The reagents in the ICAM-1 ELISA kit were placed at room temperature, and the test sample and standard were each replicated twice, then the obtained tissue supernatant and standard were fully mixed at room temperature after centrifugation, and the concentrated washing solution in the ELISA kit was diluted with distilled water, about 10 times. Determine the number of micropores required during detection, then label one by one (in addition, one hole needs to be left as a control). Add the obtained supernatant, quality control serum and 100 μl standard to the labeled micropores in turn, cover the sheet and react at 37°C for 30 min. Then pour out the solution in the micropore plate, and wash it with washing solution for 5 times, about 300 μl of washing solution is used for each micropore plate; ensure that the washing is clean, and try to absorb the residual liquid as much as possible. Then add enzyme-labeled reagent to the micropore plate, mix well, and react at 37°C for 30 min, then pour out the liquid in the micropore plate, and wash it with washing solution for 5 times again. Add color developing substrates A and B about 100 μl to each micropore plate in turn, incubate at 37°C for 10 min in the dark, and add 2 mol / L H2SO4 0.05 ml of termination liquid to terminate the reaction. Within 15 min, measure the absorbance (OD value) at 450 nm wavelength by using an enzyme-labeled instrument, take the ICAM-1 standard concentration as the abscissa, and take the sample absorbance / standard "0" point absorbance as the ordinate, draw the standard curve on the logarithmic coordinate paper, and then calculate the value of ICAM-1 in the measured sample on the standard curve.

[0072] The detection process of IL-6, MCP-1 and BAX in bladder tissue is similar to that of ICAM-1.

[0073] In the pathological environment of interstitial cystitis, the expression of IL-6 will continue to abnormally increase, and it will change from a protective factor to a destructive factor. Figure 8It can be seen that the expression of IL-6 in the model group is significantly higher than that in the blank group, while the expression of IL-6 in the TPL-exo treatment group is significantly lower and close to that in the blank group. It can be seen that TPL-exo treatment can significantly inhibit the expression of destructive factor IL-6, thereby inhibiting the inflammatory response.

[0074] In the complex pathogenesis of interstitial cystitis (IC), monocyte chemotactic protein-1 (MCP-1 / CCL2) is a crucial chemotactic factor that mainly plays the role of "recruiter" and is one of the core molecules that initiate and maintain bladder inflammation. In healthy bladder, the expression level of MCP-1 is very low. However, when the bladder epithelium is damaged, bladder wall cells (such as epithelial cells, fibroblasts) and existing immune cells will be activated to produce and release a large amount of MCP-1. Therefore, determining the expression level of MCP-1 in bladder tissue can determine the degree of damage to bladder tissue, Figure 8 It can be seen that the expression of MCP-1 in the blank group, that is, the normal healthy bladder tissue, is very low, while the expression of MCP-1 in the model group is significantly higher than that in the blank group, and TPL-exo treatment can significantly inhibit the expression of MCP-1. This plays a key role in relieving inflammatory response, increasing the threshold of bladder to pain, and inhibiting fibrosis of bladder tissue.

[0075] In interstitial cystitis (IC), initial bladder epithelial damage or dysfunction will prompt local cells to release pro-inflammatory cytokines, which will strongly stimulate bladder vascular endothelial cells and uroepithelial cells to highly express ICAM-1. The flowing immune cells in the blood recognize these highly expressed ICAM-1 through receptors on their surfaces and are tightly grabbed. The captured immune cells will then change their morphology, penetrate out of the intercellular space of the vascular endothelial cells, and migrate into the bladder interstitial tissue. After these infiltrating immune cells (such as macrophages, T cells) are activated, they will release more inflammatory factors and destructive enzymes, leading to a cascade amplification of inflammatory response and continuous destruction of bladder tissue. As can be seen, inhibiting the expression of ICAM-1 and avoiding the recognition of ICAM-1 by flowing immune cells can avoid the amplification of inflammatory response and the continuous destruction of bladder tissue, thereby Figure 8 It can be seen that the expression of ICAM-1 in the blank group is very low, and the expression of ICAM-1 in the model group is significantly increased, which leads to a cascade amplification of inflammatory response and a continuous destructive effect on the bladder in the model group; while TPL-exo treatment can significantly reduce the expression of ICAM-1, so that the expression of ICAM-1 is close to that in the blank group, thereby greatly relieving the inflammatory response of the animal and the continuous destruction of the bladder tissue.

[0076] In a healthy bladder, epithelial cells are replaced normally, and apoptosis and proliferation are in balance. In IC, this balance is broken, and the BAX-mediated apoptosis pathway is abnormally activated. A large number of bladder epithelial cells undergo programmed death and fall off. Because the cell death rate exceeds the regeneration rate, the bladder lining becomes weak and has "leakage", and cannot effectively maintain its vital barrier function (GAG layer). This further exacerbates the penetration of urine toxins, forming a vicious cycle of "injury-apoptosis-reinjury". Figure 8 It can be seen that the expression of BAX in the blank group is low, and the expression of BAX in the model group is significantly increased, and TPL-exo treatment can significantly reduce the expression of BAX, thereby greatly inhibiting the vicious cycle of "injury-apoptosis-reinjury" and relieving interstitial cystitis.

[0077] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A nano-co-delivery drug delivery carrier loaded with triptolide, characterized in that, The carrier was prepared by mixing equal amounts of umbilical cord mesenchymal stem cell exosomes and triptolide.

2. The nano-co-delivery drug delivery carrier loaded with triptolide according to claim 1, characterized in that, The particle size of the nano-co-delivery drug carrier loaded with triptolide is 72~91 nm.

3. The nano-co-delivery drug delivery carrier loaded with triptolide according to claim 1 or 2, characterized in that, The dosage of triptolide is 25 ng / mL.

4. The use of the nano-co-delivery carrier loaded with triptolide according to any one of claims 1 to 3 in the preparation of a medicament for treating and / or alleviating interstitial cystitis.

5. The use of the triptolide-loaded nano-co-delivery drug carrier according to any one of claims 1 to 3 in the preparation of a medicament for relieving and / or inhibiting inflammation of interstitial cystitis.

6. The application according to claim 5, characterized in that, The nano-co-delivery drug delivery carrier loaded with triptolide inhibits the expression of IL-6, MCP-1, ICAM-1 and BAX in inflammation.

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