Application of novel nano drug-loading delivery system of exosome-loaded ginsenoside Rh2

Abstract

The invention is applicable to the technical field of biological pharmacy, and provides application of a novel nano drug-loading delivery system of exosome-loaded ginsenoside Rh2. The ginsenoside Rh2 and the MSC-derived exosome are subjected to blending ultrasonic treatment to realize exosome drug loading, and a novel Rh2-coated MSC-exo nano drug loading delivery system is synthesized. Animal experiments show that the Rh2 (at) MSC-exo novel nano drug-loading delivery system can be targeted to ectopic intimal lesions and has an inhibition effect; the expression level of an M1 type macrophage marker CD86 is enhanced, and the expression level of an M2 type macrophage marker CD206 is inhibited; the m6A level and the METTL3 expression level of ectopic endometrial tissues are reduced; according to the present invention, RNA-seq and MIP-seq sequencing is adopted to determine that WNT2 is the potential target of the Rh2 (at) MSC-exo novel nanometer drug loading delivery system affecting ectopic inner membrane m6A modification;

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to the application of a novel nano drug delivery system of exosomes loaded with ginsenoside Rh2. Background Art

[0002] In recent years, due to environmental factors and changes in lifestyle, the incidence of endometriosis in women has shown a clear upward trend. Due to the complexity and diversity of pathogenic factors, the specific mechanism is still unclear. At present, there is no clear therapeutic target for the clinical treatment of endometriosis, so the clinical efficacy is poor. Based on a series of serious consequences caused by endometriosis, exploring the pathogenesis of endometriosis and seeking new targets for its prevention and treatment are medical problems that need to be solved urgently. Endometriosis is an estrogen-dependent disease that catalyzes androstenedione and testosterone into estradiol and estradiol, thereby promoting the production of estrogen in endometriotic lesions and eutopic endometrial tissue. Fibrotic lesions caused by changes in macrophage polarization in endometrial tissue play a role in the key steps of endometriotic lesion formation. Studies have found that endometrial fibrosis plays an important role in promoting the development and recurrence of lesions. In fibrotic lesions, M2 macrophages are abnormally infiltrated, pro-fibrotic cytokines and angiogenic cytokines are released, changes in the expression levels of m6A-related enzymes are changed, and the Wnt signaling pathway is activated. These results suggest that fibrosis may occur in endometriosis, and m6A methylation and Wnt signaling pathway may be key factors in the occurrence of fibrosis in ectopic lesions.

[0003] Ginsenoside Rh2 is a rare active ingredient of ginseng, which has anti-inflammatory and anti-cancer effects. It can effectively reduce the size of endometriotic lesions in rat peritoneum and has good pharmacological effects on endometriosis, but it has poor water solubility and low bioavailability. MSC has a significant therapeutic effect on diseases such as inflammation. In recent years, studies on MSC have confirmed that its exosomes can achieve targeted delivery of functional transmitters and have higher biosafety. The use of exosomes as carriers for treatment has received widespread attention in recent years and has high clinical application value. Considering the biosafety risk of stem cell proliferation in vivo, exosomes are not only safer, but can also interfere with the size of endometrial lesions through RNA. The combination of exosomes with the traditional Chinese medicine ingredient ginsenoside Rh2 and the synergistic treatment of endometriosis and related mechanisms are worthy of in-depth study. For this reason, the application of a new nano drug delivery system loaded with ginsenoside Rh2 is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a novel nano-drug delivery system of exosomes loaded with ginsenoside Rh2, aiming to solve the problems raised in the above-mentioned background technology.

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

[0006] Application of the new Rh2@MSC-exo nanodrug delivery system in the preparation of drugs for the treatment of endometriosis.

[0007] Furthermore, the novel Rh2@MSC-exo nano-drug delivery system can target ectopic endometrial lesions and inhibit inflammation of ectopic endometrial tissue.

[0008] Furthermore, the novel Rh2@MSC-exo nano-drug delivery system can reduce the level of endometrial fibrosis and affect macrophage polarization, thereby inhibiting the polarization of M2 macrophages by increasing the expression level of M1 macrophage marker CD86 and decreasing the expression level of M2 macrophage marker CD206.

[0009] Furthermore, the novel Rh2@MSC-exo nano-drug delivery system can reduce the m6A level and METTL3 expression level in ectopic endometrial tissue.

[0010] Furthermore, the novel Rh2@MSC-exo nano-drug delivery system can reduce the mRNA expression levels of endometrial-related genes WNT2, WNT4 and β-catenin.

[0011] Furthermore, the novel Rh2@MSC-exo nano-drug delivery system inhibits the polarization of M2 macrophages by regulating the m6A modification of WNT2 in the Wnt pathway.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention realizes exosome drug loading by ultrasonically blending ginsenoside Rh2 with MSC-derived exosomes (MSC-exo), and synthesizes a novel nano drug delivery system in which exosomes encapsulate ginsenoside Rh2, namely, the Rh2@MSC-exo novel nano drug delivery system. Animal experiments show that the Rh2@MSC-exo novel nano drug delivery system has an inhibitory effect on ectopic endometrial tissue inflammation; it can reduce the expression level of inflammatory factors (TNF-α, IL-1β, IL-6, IL-8, IL-17) in the serum of female rats, and significantly downregulate the expression level of VEGF, CA125, and EMAb in the serum; it can enhance the expression level of M1 macrophage marker CD86, inhibit the expression level of M2 macrophage marker CD206, inhibit endometrial fibrosis, and inhibit the m6A level of ectopic endometrial tissue, and METTL3 is significantly downregulated; it can reduce the mRNA expression level of endometrial-related genes WNT2, WNT4, and β-catenin. The present invention provides a solid theoretical basis and technical support for the clinical application of the novel Rh2@MSC-exo nano-drug delivery system, and provides new ideas and new targets for the prevention and treatment of endometriosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Characterization of MSCs; A is a representative image of osteogenic differentiation of MSCs, scale bar = 50 μm; B is a representative image of lipogenic differentiation of MSCs, scale bar = 50 μm; C is a representative image of chondrogenic differentiation of MSCs, scale bar = 100 μm; D: blank control (Control); CD105 positive; CD73 and CD90 positive; CD11b, CD19, CD34, CD45 and HLA-DR negative.

[0015] Figure 2 Characterization of MSC-exo; A is a representative transmission electron microscopy (TEM) image of MSC-exo, scale bar = 1 μm; B is a representative transmission electron microscopy (TEM) image of MSC-exo, scale bar = 200 nm; C is Western Blot identification of MSC-exo surface markers.

[0016] Figure 3 This is the morphological observation of ectopic endometrial tissue in rats in different treatment groups.

[0017] Figure 4 The expression of serum inflammatory factors TNF-α, IL-1β, IL-6, IL-8, IL-17, CA125, EMAb and VEGF in rats a P<0.05, vs Control group; b P<0.05, vs EMs group.

[0018] Figure 5 Immunofluorescence was used to detect the expression of M1 macrophage marker CD86 and M2 macrophage marker CD206 in normal and ectopic endometrial tissues of rats. DAPI (4',6-diamidino-2-phenylindole) was used to mark the position of the cell nucleus, and Merge showed the relative position relationship between the fluorescence signals of M1 macrophage marker CD86 and M2 macrophage CD206 and the cell nucleus.

[0019] Figure 6 RT-qPCR was used to detect the effect of Rh2@MSC-exo on m6A levels and related enzymes.

[0020] Figure 7 RT-qPCR was used to detect the effect of Rh2@MSC-exo on the expression of Wnt pathway-related genes in ectopic endometrial tissue.

[0021] Figure 8 It is a mechanism diagram of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0024] Example 1: Drug preparation, experimental animals and tissue sample collection;

[0025] 1) Preparation of a novel nano-drug delivery system of exosomes loaded with ginsenoside Rh2, i.e., Rh2@MSC-exo novel nano-drug delivery system;

[0026] The rare ginsenoside Rh2 was extracted and separated from Changbai Mountain ginseng, and MSC-derived exosomes were extracted at the same time. The exosomes were mixed and ultrasonically loaded to achieve drug delivery, and a new nano-drug delivery system with exosomes encapsulating ginsenoside Rh2 was synthesized.

[0027] 1.1 Preparation of MSCs (mesenchymal stem cells);

[0028] First, umbilical cord tissue was separated (from full-term healthy newborns in the Third Hospital of Jilin University, voluntarily donated after informed consent from their parents. The donors were in good maternal and fetal health and met the collection criteria), rinsed three times with phosphate-buffered saline (PBS, Sigma, Germany), and then the umbilical cord was cut into tissue blocks. The cut tissue blocks were placed in a 100mm culture dish (Corning, USA), with each tissue block separated by about 3mm. 8-10ml of DMEM medium (stem cells, Canada) containing 20% ​​fetal bovine serum (FBS, HyClone, USA) was added to the culture dish. The culture dish was placed in a constant temperature carbon dioxide incubator (ThermoFisher, USA) and cultured at 37°C, and the DMEM medium was replaced every 3 days. When the cells around the tissue grow to 80%, cell passage can be performed. All experimental procedures used MSCs of the 3rd to 6th generations, which were subsequently cultured in HyMEM medium (HyClone, USA) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0029] 1.2 MSCs identification;

[0030] MSCs were cultured in mesenchymal stem cell adipogenic differentiation medium (Sigma, Germany) for 18-21 days, and then the cells were stained with Oil Red Stain (Solarbio, China), observed and photographed under a microscope.

[0031] MSCs were cultured in mesenchymal stem cell osteogenic differentiation medium (Sigma, Germany) for 28 days and then stained with Alizarin Red (Solarbio, China) for 20-30 min, after which the cells were examined under a microscope and photographed.

[0032] To form chondrocytes, MSCs were cultured in mesenchymal stem cell chondrogenic differentiation medium (Sigma, Germany) for 21 days. Chondrocytes were sliced ​​and then stained with Alcian blue (Solabio, China) for 30 min, washed 3 times with water, and then restained with nuclear solid red for 5 min, and observed and examined under a microscope.

[0033] Flow cytometric analysis: To evaluate the phenotypic profile of MSCs for cell characterization, we used antibodies against specific cell surface markers (CD90, CD105, CD73, CD11b, CD19, CD34, CD45, and HLA-DR) from Biogen, USA.

[0034] Results: Characterization of MSCs;

[0035] The three-lineage differentiation potential determines the differentiation potential of MSCs. Red calcium nodules can be seen after MSCs osteogenic differentiation ( Figure 1 Middle A). After inducing MSCs to differentiate into adipocytes, intracellular lipid droplets can be seen ( Figure 1 In addition, the cartilage spheroids were sliced ​​and stained with Alisin blue to show blue ( Figure 1 C). Flow cytometry analysis showed ( Figure 1 (D), compared with the blank group, MSCs expressed CD90, CD105 and CD73, but did not express CD11b, CD19, CD34, CD45 and HLA-DR.

[0036] 1.3 Isolation and identification of MSC-exo;

[0037] All experiments used cells of passage 3-6, and their culture supernatants were collected. The specific steps were as follows: first, the supernatant was centrifuged at 300g for 10min to remove dead cells. Then, the supernatant was centrifuged at 2000g for 10min to remove cell debris. After that, the supernatant was centrifuged at 10000g for 1h to precipitate exosomes. Finally, the precipitate was resuspended in phosphate-buffered saline (PBS) and centrifuged at 100000g for 90min to remove soluble and secreted proteins, and the precipitate was collected as exosomes. The size and concentration of exosomes were determined using "nanoparticle" tracking analysis (NTA, NanoSight LM10, UK), and each exosome sample was tested 5 times. Morphological examination was performed by an 80kV transmission electron microscope (TEM, Hitachi, Japan) to observe the specific morphology of exosomes.

[0038] 1.4 Western Blot method;

[0039] To verify the presence of exosomes, Western Blot was used to detect exosome markers, including CD9 (1:1000, Proteintech, China), TSG101 (1:1000, Proteintech, China), CD81 (1:1000, Proteintech, China) and Calnnexin (1:1000, Proteintech, China). The specific steps were as follows: MSC-exo was lysed in lysis buffer (RIPA:PMSF=99:1, Abcam, USA), centrifuged at 16000g for 15min, and the supernatant was taken as a protein sample. The protein concentration in the sample was determined using the HBCA detection kit (Beyotime, China), and the protein was denatured by heating at 95°C for 5min. The denatured protein was separated by SDS-PAGE electrophoresis, and then the target protein was transferred to NC membrane (Millipur, Germany), and the NC membrane was dried after soaking in skim milk powder at room temperature for 1h. The NC membranes were treated with CD9- and Calnexin-specific antibodies at 4°C overnight, and after washing three times with TBST, the NC membranes were incubated with secondary antibody diluent (1:5000, Proteintech, China) at room temperature for 1 h.

[0040] Results: Characterization of MSC-exo;

[0041] MSC-exo was isolated from the cultured cell supernatant. TEM images showed that MSC-exo was a double-layer biomembrane structure ( Figure 2 Middle A). Lower magnification shows uniform distribution of exosomes of different sizes ( Figure 2 (B). Immunoblotting of exosome markers showed that the prepared MSC-exo expressed the exosome characteristic marker CD9, but did not express Calnexin ( Figure 2 Middle C).

[0042] 2) Animal grouping and treatment;

[0043] Sixty 2-month-old SD female rats (Beijing Weitonglihua Experimental Animal Technology Co., Ltd., OR1001) were selected and randomly divided into six groups, with 10 rats in each group. The groups were: control group (Control group), endometriosis model group (normal saline treatment group, i.e. EMs model group; new nano drug delivery system treatment group, i.e. Rh2@MSC-exo treatment group).

[0044] Animal modeling: female rats in estrus were taken, anesthetized with isoflurane, fixed in the supine position, and the abdomen was trimmed and the skin was disinfected. An incision of about 1.5 cm long was made in the upper left middle of the lower abdomen, the left uterine horn was picked out, the mesenteric vessels were ligated, and then the two ends of the uterine segment to be removed were ligated, and a 1.5-2.0 cm long uterine tissue was cut. The endometrium was separated in sterile PBS. A 1cm×1cm endometrial fragment was cut, and the endometrium was facing the abdominal wall and sutured to the inner side of the anterior abdominal wall with sutures. The skin was sutured and the rats were raised normally. All rats used for modeling successfully formed ectopic foci, proving that the modeling was successful. The drug was administered by tail vein injection, once every 7 days for four weeks, for a total of 5 times. Blood was collected after the treatment, and uterine tissue samples were taken for subsequent analysis.

[0045] The experimental animals were adapted to feeding for one week before the experiment. The feeding environment was maintained with a 12-hour day and night alternation, the temperature was controlled at (23±1)℃, and the humidity was controlled at (55±5)%. The rats were free to take in food and water. During the experiment, the rats were weighed daily, and the amount of food and water consumed was recorded; the mental state, activity, fur changes, secretions, and whether there was death of the rats were observed.

[0046] Example 2, determination indicators and methods;

[0047] 1) Histopathological observation;

[0048] The uterine tissue was fixed in 10% neutral formalin solution, routinely rinsed for 72 hours, dehydrated and transparent, paraffin embedded, 4-6 μm tissue sections, baked at 60-65°C for 30 minutes for dewaxing, stained with hematoxylin-eosin (HE), sealed, and histopathological changes were observed under a microscope.

[0049] Results: Morphological observation of rat ectopic endometrium;

[0050] After modeling, abdominal ectopic lesions were observed by small animal ultrasound ( Figure 3 ), laparotomy revealed that the ectopic lesions were located on the abdominal wall and were transparent cystic or red nodular ( Figure 3(middle EMs).

[0051] After injection treatment, the changes of ectopic foci in the rats' abdominal cavity were recorded before and after treatment. Figure 3 Middle Con: normal rat smooth endometrium; Figure 3 Middle EMs: ectopic endothelial cells, which are transparent cystic or red nodular; Figure 3 Medium Rh2: The lesions in the Rh2 treatment group were slightly reduced; Figure 3 Middle MSC-exo: The lesions in the MSC-exo treatment group were slightly reduced; Figure 3 Rh2@MSC-exo: The lesions in the Rh2@MSC-exo treatment group were significantly reduced.

[0052] Morphological observation of ectopic endometrium revealed that Rh2, MSC-exo, and Rh2@MSC-exo all reduced the size of ectopic lesions and had an inhibitory effect on EMs, and the Rh2@MSC-exo treatment group had the most significant effect. This suggests that the new Rh2@MSC-exo nano-drug delivery system can target ectopic endometrial lesions and inhibit inflammation of ectopic endometrial tissue.

[0053] 2) Detection of the expression of endometrial macrophage-related markers: Immunofluorescence method was used to determine the expression levels of related proteins;

[0054] Results: Expression of rat endometrial macrophage-related markers;

[0055] Immunofluorescence: Immunofluorescence was used to detect the expression of M1 macrophage markers CD86 and M2 macrophages CD206 in rats. Figure 5 As shown in the figure, compared with the control group, the expression of M1 macrophage marker CD86 in rats in the EMs model group decreased, while the expression of M2 macrophage CD206 increased. After intervention with the new Rh2@MSC-exo nano drug delivery system, the expression of M1 macrophage marker CD86 increased, while the expression of M2 macrophage CD206 decreased, that is, Rh2@MSC-exo can inhibit the polarization of M2 macrophages, further leading to a decrease in pro-fibrotic factors and pro-angiogenic factors, and ultimately inhibiting endometrial fibrosis, achieving a therapeutic effect on EMs.

[0056] 2) Determination of serum related factor levels;

[0057] Enzyme-Linked ImmunoSorbent Assay (ELISA) was used to detect the expression levels of TNF-α (tumor necrosis factor-α), IL-1β (interleukin-1β), IL-6 (interleukin-6), IL-8 (interleukin-8), and IL-17 (interleukin-17) in serum. At the same time, the expression levels of CA125 (carbohydrate antigen 125), EMAb (anti-epidermal growth factor receptor EGFR monoclonal antibody), and VEGF (vascular endothelial growth factor) in serum were detected.

[0058] like Figure 4 As shown in the figure, ELISA results showed that the EMs model group could upregulate the expression of inflammatory factors (TNF-α, IL-1β, IL-6, IL-8, IL-17) in the serum of female rats, and upregulate the expression levels of CA125, EMAb and VEGF in the serum. The Rh2@MSC-exo treatment group could downregulate the expression of inflammatory factors (TNF-α, IL-1β, IL-6, IL-8, IL-17) in the serum of female rats, and downregulate the expression levels of CA125, EMAb and VEGF in the serum. CA125, EMAb and VEGF are helpful for the early diagnosis of endometriosis, suggesting that the new Rh2@MSC-exo nano-drug delivery system may treat endometriosis by inhibiting inflammatory response.

[0059] 3) Detection of mRNA expression levels of ectopic endometrial m6A-related factors (METTL3, METTL14, ALKBH, YTHDF1, YTHDF2, YTHDF3, FTO, WTAP); Real-time fluorescence quantitative PCR (RT-qPCR) was used to determine the expression levels of related enzyme factors.

[0060] Results: Expression of m6A-related enzyme molecules in the endometrium of rats in each group;

[0061] like Figure 6As shown in the figure, the results of RT-qPCR detection of m6A level and expression of key m6A enzyme molecules in the uterus showed that compared with the control group, the m6A level in the EMs model group increased, and after intervention with the new Rh2@MSC-exo nano-drug delivery system, the m6A level decreased, indicating that the new Rh2@MSC-exo nano-drug delivery system can inhibit the m6A level of ectopic endometrial tissue. Compared with the control group, the expression levels of endometrial-related genes METTL3 and YTHDF1 in the EMs model group were significantly upregulated, that is, the m6A methylation modification level in endometrial macrophages in the EMs model group was significantly increased, and after intervention with the new Rh2@MSC-exo nano-drug delivery system, the expression levels of METTL3 and YTHDF1 were significantly decreased, that is, the m6A methylation modification level in endometrial macrophages was significantly decreased after intervention with the new Rh2@MSC-exo nano-drug delivery system.

[0062] 4) Detection of mRNA expression levels of endometrial Wnt pathway-related genes (WNT2, WNT4 and β-catenin); Real-time fluorescence quantitative PCR was used to determine the expression levels of related genes;

[0063] Results: Expression of genes related to the Wnt pathway in the endometrium of rats in each group;

[0064] like Figure 7 As shown in the figure, the results of RT-qPCR detection of key uterine gene expression showed that compared with the control group, the mRNA expression levels of endometrial-related genes WNT2, WNT4 and β-catenin in the EMs model group were significantly upregulated. After intervention with the Rh2@MSC-exo novel nano-drug delivery system, the mRNA expression levels of the above genes were significantly decreased (P < 0.05), indicating that the Rh2@MSC-exo novel nano-drug delivery system may have the effect of regulating the expression of genes related to the Wnt pathway.

[0065] To further explore this mechanism, we used RNA-seq sequencing to observe the presence of the Wnt pathway in the endometrium of surgically modeled rats, and further screened out corresponding changes in the m6A level of WNT2 in the Wnt pathway through MIP-seq sequencing. This finding suggests that WNT2 may be a potential target for the new Rh2@MSC-exo nanodrug delivery system to affect the m6A modification of the ectopic endometrium.

[0066] Based on the above sequencing results, we hypothesized that the new Rh2@MSC-exo nano-drug delivery system may inhibit the polarization of M2 macrophages by regulating the m6A modification of WNT2 in the Wnt pathway of ectopic macrophages, thereby reducing the occurrence of endometriosis. This hypothesis was supported by experimental data, that is, the mRNA expression levels of endometrial-related factors (including ectopic endometrial m6A-related factors and endometrial Wnt pathway-related genes) in rats in the EMs model group were significantly higher than those in the control group and the Rh2@MSC-exo treatment group (P<0.05).

[0067] In the above experiments, quality control: quality control was used to reduce the influence of some interfering factors on the experimental results. The microscopic observation of the slices was carried out by blind film method; the order of measuring the expression levels of related genes and proteins in each group of rats was randomly determined. Statistical analysis: The experimental data were all normally distributed. The data were expressed in the form of SPSS 22.0 statistical software. For comparison among multiple groups, the Student-Newman-Keuls (SNK) test was used when the variances were equal; the Kruskal-Wallis (Kruskal-Wallis) test was used when the variances were unequal. P < 0.05 was considered statistically significant.

[0068] in conclusion:

[0069] 1. If Figure 8 As shown, the present invention uses MSC-exo and ginsenoside Rh2 to construct a new Rh2@MSC-exo nano-drug delivery system, which is injected into the EMs model group for in vivo treatment.

[0070] 2. HE results showed that the Rh2@MSC-exo group could target ectopic endometrial lesions and inhibit the inflammation of ectopic endometrial tissue.

[0071] 3. Immunofluorescence results showed that Rh2@MSC-exo could inhibit the polarization of M2 macrophages.

[0072] 4. Compared with the control group, the m6A methylation modification level in endometrial macrophages in the EMs model group was significantly increased, and the METTL3 content was significantly upregulated. After intervention with the Rh2@MSC-exo novel nanodrug delivery system, the expression level of METTL3 was significantly decreased.

[0073] 5. Compared with the control group, the mRNA expression levels of endometrium-related genes WNT2, WNT4 and β-catenin in the EMs model group were significantly upregulated. After intervention with the new Rh2@MSC-exo nano-drug delivery system, the mRNA expression levels of the above genes were significantly decreased.

[0074] 6. The mRNA and protein expression levels of endometrium-related factors in the EMs model group were significantly higher than those in the control group and the Rh2@MSC-exo treatment group (P<0.05). This suggests that Rh2@MSC-exo may inhibit the polarization of M2 macrophages by regulating the m6A modification of WNT2 in the WNT2 pathway, thereby reducing the occurrence of endometriosis.

[0075] In summary, the present invention provides a solid theoretical basis and technical support for the clinical application of the new Rh2@MSC-exo nanodrug delivery system, and provides new ideas and new targets for the prevention and treatment of endometriosis.

[0076] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. Application of the new Rh2@MSC-exo nano-drug delivery system in the preparation of drugs for the treatment of endometriosis.

2. The use according to claim 1, characterized in that: The novel Rh2@MSC-exo nano-drug delivery system can target ectopic endometrial lesions and inhibit inflammation of ectopic endometrial tissue.

3. The use according to claim 1, characterized in that: The novel Rh2@MSC-exo nano-drug delivery system inhibits the polarization of M2 macrophages by increasing the expression level of M1 macrophage marker CD86 and decreasing the expression level of M2 macrophage marker CD206.

4. The use according to claim 1, characterized in that: The novel Rh2@MSC-exo nano-drug delivery system can reduce the m6A level and METTL3 expression level of ectopic endometrial tissue.

5. The use according to claim 1, characterized in that: The novel Rh2@MSC-exo nano-drug delivery system can reduce the mRNA expression levels of endometrial-related genes WNT2, WNT4 and β-catenin.

6. The use according to claim 1, characterized in that: The novel Rh2@MSC-exo nano-drug delivery system inhibits the polarization of M2 macrophages by regulating the m6A modification of WNT2 in the Wnt pathway.

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