Application of C3 and / or C3a and / or C3aR as target spot in preparation of endometriosis related diagnosis and / or treatment preparation

Abstract

The invention discloses application of C3 and / or C3a and / or C3aR as a target spot in preparation of a preparation for diagnosing and / or treating diseases related to endometriosis, relates to the technical field of biological medicines, and provides application of C3 and / or C3a and / or C3aR as a target spot in preparation of a preparation for diagnosing and / or treating diseases related to endometriosis. The endometriosis comprises a uterus-sacral ligament type endometriosis, a peritoneal type endometriosis, a rectum-vagina type endometriosis, a deep infiltration type endometriosis, an ovarian type endometriosis, an abdominal wall incision type endometriosis, a perineum incision type endometriosis and a lung type endometriosis; and at least one of nasal endometriosis. The endometriosis can be diagnosed by detecting the content of at least one of C3, C3a and C3aR; the C3 and / or C3a and / or C3aR inhibitor can be used for reducing the focus and treating the endometriosis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to the use of C3 and / or C3a and / or C3aR as targets in the preparation of diagnostic and / or therapeutic agents for endometriosis-related diseases. Background Technology

[0002] Endometriosis (EM) is a common benign gynecological disease in women of reproductive age, characterized by the colonization and growth of endometrial-like tissue outside the uterus. Clinical manifestations of endometriosis mainly include pain, decreased fertility, and menstrual abnormalities, severely impacting the quality of life of women of reproductive age.

[0003] Currently, medications for treating endometriosis (EM) include dinogest, oral contraceptives, and gonadotropin-releasing hormone agonists. These medications work by affecting estrogen and progesterone levels, thus shrinking or slowing the growth of lesions. However, the use of hormonal medications can cause side effects such as hot flashes, night sweats, osteoporosis, and irregular vaginal bleeding, impacting patients' quality of life.

[0004] Therefore, how to develop drugs that target non-hormonal targets, avoid changes in patients' hormone levels caused by hormone drugs, and reduce the side effects caused by hormone drug treatment are technical problems that need to be solved. Summary of the Invention

[0005] Currently, the main drugs used to treat endometriosis are hormonal medications, which can easily cause fluctuations in hormone levels in patients, leading to pseudopregnancy, temporary amenorrhea, and side effects such as nausea, weight gain, irregular vaginal bleeding, hot flashes, night sweats, and osteoporosis. To address the issues of hormone fluctuations and side effects caused by hormonal drug treatment for endometriosis, this invention provides the application of C3, C3a, and C3aR as targets in the preparation of diagnostic and / or therapeutic agents related to endometriosis. The applicant unexpectedly discovered that complement C3 and its cleavage product C3a, along with its receptor C3aR, are significantly elevated in ectopic endometrial tissue; therefore, C3, C3a, and C3aR can serve as diagnostic biomarkers for endometriosis. By intraperitoneally injecting C3 and / or C3a and / or C3aR inhibitors into EM model mice, C3 and C3aR are targeted and blocked, thereby inhibiting the C3a-C3aR interaction, which in turn inhibits the ability of macrophages to enhance endometrial stroma migration, leading to lesion shrinkage. This suggests that C3, C3a, and C3aR could serve as novel intervention targets for the clinical treatment of endometriosis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of C3 and / or C3a and / or C3aR as targets in the preparation of diagnostic and / or therapeutic agents for endometriosis-related diseases.

[0008] Secondly, the present invention provides the application of C3 and / or C3a and / or C3aR targets as diagnostic biomarkers in the preparation of reagents or kits for diagnosing endometriosis.

[0009] Furthermore, the endometriosis includes at least one of the following: uterosacral ligament type endometriosis, peritoneal type endometriosis, rectovaginal type endometriosis, deep infiltrative type endometriosis, ovarian type endometriosis, abdominal wall incision type endometriosis, perineal incision type endometriosis, pulmonary type endometriosis, and nasal cavity type endometriosis.

[0010] Thirdly, the present invention provides the use of reagents for detecting C3 and / or C3a and / or C3aR in the preparation of kits for diagnosing diseases caused by endometriosis.

[0011] Furthermore, the reagents for detecting C3 and / or C3a and / or C3aR include at least one of PCR primers, qPCR primers, FISH nucleic acid probes, antibodies, and binding peptides.

[0012] Fourthly, the present invention provides the use of inhibitors of C3 and / or C3a and / or C3aR in the preparation of medicaments for treating endometriosis.

[0013] Furthermore, the inhibitor is one of gRNA, dsRNA, siRNA, shRNA, or microRNA that targets the DNA and / or RNA of C3 and / or C3aR to inhibit gene transcription and protein expression; the inhibitor is a targeted cell that targets the DNA sequence of C3aR, through in vitro gene modification and amplification of autologous cells, and then reinfused.

[0014] Further, the inhibitor is a biological or chemical agent that targets the protein sequence of C3 and / or C3a and / or C3aR and has an inhibitory or antagonistic effect on C3 and / or C3a and / or C3aR; the biological or chemical agent includes at least one of antibodies, small molecule peptide compounds, and protein degradation-targeting chimeras.

[0015] Furthermore, the inhibitor is SB290157, with the chemical formula shown below.

[0016]

[0017] Fifthly, the present invention provides a diagnostic kit for endometriosis, comprising C3 and / or C3a and / or C3aR targets as diagnostic biomarkers.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. Compared with eutopic endometrial tissue, C3 and / or C3a and / or C3aR are significantly elevated in ectopic endometrial tissue. Therefore, endometriosis can be diagnosed by detecting the content of any one of C3, C3a, and C3aR alone, or by detecting the content of any two or three of C3, C3a, and C3aR.

[0020] 2. By using small molecule compounds such as peptides and neutralizing antibodies to competitively bind to C3aR, the interaction between C3a and C3aR can be blocked. Alternatively, gRNA, dsRNA, siRNA, shRNA, microRNA, etc., can be used to target the RNA or DNA of C3aR or C3 to block the generation of C3aR or C3 DNA and RNA. Alternatively, autologous cells can be genetically modified and amplified in vitro and reinfused into targeted cells using the DNA sequence of C3aR as the target. Alternatively, protein degradation targeting chimeras (PROTAC) can be prepared by combining C3a or C3aR targets to promote the degradation of C3a or C3aR, thereby blocking the C3a-C3aR interaction and achieving targeted blocking of C3 and / or C3a and / or C3aR. This can effectively inhibit lesion growth, reduce the volume and weight of lesions, and achieve the effect of treating endometriosis.

[0021] 3. C3 and / or C3a and / or C3aR inhibitors can reduce the ability of macrophages to migrate to endometrial stromal cells, effectively reduce the adhesion and invasion of endometrium outside the uterus, inhibit the migration of ectopic endometrial stromal cells in existing lesions to the surrounding lesions, thereby achieving the effect of inhibiting the spread of lesions.

[0022] 4. C3 and / or C3a and / or C3aR inhibitors can significantly increase granzyme B expression, enhance the killing function of CD8 positive T cells, and CD8 positive T cells can kill ectopic endometrial stromal cells, which in turn facilitates apoptosis of ectopic endometrial stromal and epithelial cells, as well as the clearance of endometrial fragments and ectopic lesions in the abdominal cavity, and inhibits lesion growth.

[0023] 5. C3 and / or C3a and / or C3aR inhibitors affect the immune microenvironment by altering the functional state of immune cells such as macrophages and CD8-positive T cells, thereby reversing the state of immunosuppression and shrinking lesions. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Single-cell transcriptome sequencing results for endometriosis;

[0026] Figure 2 Immunofluorescence representations of ectopic (EM-ec) and eutopic endometrial tissues (EM-eu) from patients with endometriosis, and eutopic endometrial tissues from the control group (NC-eu); where GFP:AF488 fluorescence indicates C3 expression in the tissues; and RFP:AF594 fluorescence indicates vimentin expression in the tissues.

[0027] Figure 3 Western blot representations and statistical figures of ectopic (EM-ec) and eutopic endometrial tissues (EM-eu) from patients with endometriosis, and eutopic endometrial tissues from the control group (NC-eu); where C3: complement protein 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; C3 expression: C3 expression level; EM-Ec group: ectopic endometrial tissue; EM-Eu group: eutopic endometrial tissue from patients with endometriosis; NC-Eu group: endometrial tissue from the control group;

[0028] Figure 4 The purpose of this study was to detect C3aR expression in ectopic (EM-ec) and eutopic endometrial tissues (EM-eu) of patients with endometriosis and in eutopic endometrial tissues of the control group (NC-eu) by flow cytometry. The percentage of C3aR expression represents the proportion of C3aR-positive cells in different cell populations.

[0029] Figure 5 The expression of C3aR in ascites cells of EM mice (EM group) and control mice (NC group); where % of C3aR expression in PF: percentage of C3aR positive cells in ascites (PF);

[0030] Figure 6 The expression of C3aR in ectopic lesions (EM-ec) and eutopic endometrium (EM-eu) of EM mice and eutopic endometrium (NC-eu) of control mice was shown; where, % of C3aR expression in immune cells: the percentage of C3aR positive immune cells in different tissues;

[0031] Figure 7 The expression of C3aR in different immune cells in ectopic lesions of EM mice; where % of C3aRexpression: percentage of different immune cells in C3aR-positive cells; macrophage: macrophage; cDC: dendritic cells; monocytes: monocytes; Neu: neutrophils; CD4+T: CD4-positive T cells; CD8+T: CD8-positive T cells; B cells: B cells; NK: natural killer cells;

[0032] Figure 8 Images and weight statistics of lesions in EM mice in the C3aR inhibitor-treated (C3aRA group) and untreated (DMSO group) groups; where Weight of the lesion: lesion weight;

[0033] Figure 9 The expression of CD206 and iNOS in ascites macrophages of EM mice in the C3aR inhibitor-treated (C3aRA group) and untreated (DMSO group) groups; where MFI ofCD206 in PF: mean fluorescence intensity of CD206 in peritoneal macrophages; MFIofinosin PF: mean fluorescence intensity of iNOS in peritoneal macrophages.

[0034] Figure 10 The expression of CD206 and iNOS in macrophages of ectopic lesions in EM mice in the C3aR inhibitor-treated (C3aRA group) and untreated (DMSO group) groups; where MFIofCD206in PF: mean fluorescence intensity of CD206-positive macrophages in ectopic lesions; MFIofinosin PF: mean fluorescence intensity of iNOS-positive macrophages in ectopic lesions.

[0035] Figure 11 The number of CD8-positive T cells in ectopic lesions (EM-ec) and eutopic endometrium (EM-eu) of EM mice in the C3aR inhibitor-treated (C3aRA group) and untreated (DMSO group) groups; where %ofCD8: percentage of CD8-positive T cells in different tissues;

[0036] Figure 12Granulase B expression in CD8-positive T cells in ascites (PF), situ endometrium (EM-eu), ectopic lesions (EM-ec), and spleen (spleen) of EM mice in C3aR inhibitor-treated (C3aRA group) and untreated (DMSO group) groups; where MFI of GZMB: mean fluorescence intensity of granase B in CD8-positive T cells in different tissues.

[0037] Figure 13 Representative figures and statistical graphs showing the effect of macrophages stimulated by C3a or C3a combined with C3aR inhibitors on the migration ability of endometrial stromal cells; where, NC group / nc: ​​control group, no treatment; C3a group / C3a: macrophage group pretreated with C3a; C3a+C3aRA group / C3a+C3aRA: macrophage group pretreated with C3a combined with C3aR inhibitors; Cell count: number of endometrial stromal cells that pass through the chamber. Detailed Implementation

[0038] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] Unless otherwise specified, the reagents, instruments, equipment, preparation methods, or processes described in the following embodiments are all commonly used techniques in the field.

[0040] Example 1: C3 expression was significantly increased in EM patients.

[0041] (1) Tissue Collection and Processing: Tissues were obtained from Shandong Provincial Hospital, and all patients signed informed consent forms. Experimental group 1 consisted of ectopic lesion tissue (i.e., ectopic endometrial tissue) from patients with endometriosis, experimental group 2 consisted of endometrial tissue from patients with endometriosis, and the control group consisted of situ endometrial tissue from patients without endometriosis, and the situ endometrial tissue consisted of healthy endometrial tissue without lesions. The tissues from experimental groups 1, 2, and the control group were washed with cold phosphate-buffered saline (PBS) and then divided into three parts. The first part of the tissue was fixed, embedded, and sectioned sequentially, the second part of the tissue was cryopreserved, and the third part of the tissue was digested into single cells.

[0042] Specifically, the procedure for the third part of the tissue was as follows: the tissue was minced with scissors, and digestion solution (basal culture medium containing 0.125% novel collagenase) was added to the tissues of experimental group 1, experimental group 2 and control group respectively. The tissues were digested at 37℃ for 45 min-1 h. The digestion was terminated with an equal volume of PBS to digest the digestion solution. The cell suspension was obtained by passing the cells through a 70 μm cell sieve, centrifuged for 5 min, the supernatant was discarded, and the cell pellet was resuspended in lysine rehydration solution. The cells were mixed, lysed on ice for 5-10 min, and then terminated with an equal volume of PBS to lysine rehydration solution. The cells were centrifuged for 5 min, the supernatant was discarded, and the cell pellet was resuspended in 98% PBS + 2% FBS (fetal bovine serum) (PBS to FBS volume ratio). Single cell suspensions were obtained for experimental group 1, experimental group 2 and control group respectively.

[0043] (2) Single-cell sequencing and analysis: The three groups of single-cell suspensions prepared in step (1) were analyzed, and the cells with viability greater than 80%, cell mass ratio less than 10%, and cell density greater than 1×10⁻⁶ were selected. 6 Single-cell libraries were constructed using single-cell suspensions at a density of / mL. Specifically, scRNA-seq libraries were constructed using the Chromium Single-Cell Kit, and cell population analysis and annotation, functional enrichment analysis, and pathway analysis were performed based on single-cell data.

[0044] The results are as follows Figure 1 As shown, the expression of C3 and C3aR in the ectopic endometrial tissue of patients with endometriosis is significantly increased, with C3 mainly expressed in fibroblast populations and C3aR mainly expressed in macrophage populations.

[0045] (3) Flow cytometry to determine C3aR content: The single-cell suspensions of experimental group 1, experimental group 2 and control group prepared in step (1) were placed in flow cytometry tubes (i.e. sample tubes). The single-cell suspensions in the three sample tubes were subjected to the following operations: First, the cell pellets in each sample tube were obtained by centrifugation. The cell pellets in the three sample tubes were resuspended with HBSS and the live / dead dye FVD was added. Apc-cy7 antibody was incubated on ice in the dark for 30 min. 1 ml of HBSS was added to each tube to stop staining, and the cells were centrifuged to obtain a pellet. Next, the cell pellet was resuspended in PBS, and surface staining was performed using CD45-APC and C3aR-PE antibodies. The cells were incubated on ice in the dark for 30 min, and 1 ml of PBS was added to each tube to stop staining. The cells were centrifuged for 5 min to obtain a pellet. Then, 200 μl of fixation and permeabilization buffer was added to each tube to resuspend the cell pellet, and the cells were incubated on ice in the dark for 30 min for fixation and permeabilization. Finally, 1 ml of washing buffer was added to each tube to stop staining, and the cells were centrifuged to obtain a pellet. The cell pellet was resuspended in washing buffer, and intracellular staining was performed using CD68-AF405 antibody. The cells were incubated on ice in the dark for 1 h, and 1 ml of washing buffer was added to each tube to stop staining. The cells were centrifuged for 5 min to obtain a pellet. 500 μl of 98% PBS + 2% FBS (volume ratio) was added to each tube to resuspend the cell pellet. The C3aR content in experimental groups 1, 2, and the control group was then measured.

[0046] The expression of C3aR in ectopic endometrial tissue and its matched eutopic endometrial tissue, as well as in the control group eutopic endometrial tissue, was analyzed by flow cytometry. The results are as follows: Figure 4 As shown, C3aR is mainly expressed on CD45 positive cells (i.e. immune cells), and the expression level of C3aR in ectopic endometrial tissue is significantly higher than that in eutopic endometrial tissue.

[0047] (4) The expression and location of C3 were observed using immunofluorescence. The following steps were performed in sequence:

[0048] Dewaxing and hydration treatment: The first part of the tissue in step (1) is dewaxed and hydrated. Specifically, the oven temperature is set to 60℃ and the slides are baked for 1.5 hours. The dewaxing and hydration are carried out in sequence through the steps in Table 1 to obtain the slide tissue.

[0049] Table 1 Dewaxing and Hydration Parameters

[0050]

[0051] Antigen retrieval: Dilute 20×EDTA antigen retrieval solution with triple-distilled water to 1×, add 1×EDTA antigen retrieval solution to the retrieval cup, microwave on high for 5 minutes to bring it to a boil, then add tissue sections to the retrieval cup, microwave on low for 15 minutes, turn off the heat and place the retrieval cup in cold water to cool to room temperature, wash with PBS 3 times, 2 minutes each time, to obtain the retrieval tissue.

[0052] Sealing: Dry the residual washing solution on the repair tissue, add goat serum, and incubate at room temperature for 30 minutes to obtain the sealed tissue.

[0053] Primary antibody incubation: Shake off the blocking solution on the tissue, dilute the primary antibody (mouse-derived vimentin antibody, 1:1000 (v / v) dilution; rabbit-derived C3 antibody, 1:200 (v / v) dilution) with goat serum, add the mixed primary antibody working solution until it completely covers the blocked tissue area, incubate at 4°C overnight, and then wash 3 times with Tris buffer (TBST) containing Tween 20, 5 min each time, to obtain the primary antibody-incubated tissue.

[0054] Secondary antibody incubation: Dilute the secondary antibody (AF488-coupled goat anti-rabbit secondary antibody, the ratio of secondary antibody to PBS is 1:200 (v / v); AF594-coupled donkey anti-mouse secondary antibody, 1:200 (v / v)) with PBS, then add mixed secondary antibody (the two secondary antibodies are mixed at a volume ratio of 1:1) working solution until it completely covers the tissue area incubated with the primary antibody, incubate at room temperature for 50 min, wash with TBST 3 times, 5 min each time, to obtain the tissue incubated with the secondary antibody.

[0055] Mounting: Add mounting medium containing DAPI (4',6-diamidino-2-phenylindole) to the surface of tissue incubated with secondary antibody, dry at 4°C in the dark, and observe the expression and location of C3 under a microscope.

[0056] Immunofluorescence analysis was performed on ectopic endometrial tissue and its matched eutopic endometrial tissue from the same patient, as well as eutopic endometrial tissue from the control group. The results are as follows: Figure 2 As shown, complement C3 is mainly expressed in ectopic endometrial stromal cells, and the C3 expression level in ectopic endometrial tissue is significantly higher than that in situ endometrium of EM patients and in situ endometrium of control patients.

[0057] (5) Using Western blotting Detection of C3 content by blot: First, prepare 10% lower and upper gels according to the kit, load 10 μl of the second part of tissue prepared in step (1), add 5 μl of protein marker on both sides, and electrophoresis at 120V for 45 min; Second, transfer membrane at 400mA constant current for 30 min, wherein the transfer clamp is assembled from the gel and methanol-activated PVDF membrane, including: black side - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - white side, the PVDF membrane is blocked with 5% skim milk powder at room temperature for 1-2 h, and washed 3 times with TBST; Third, incubate primary antibody (C3, 1:5000; GAPDH: 1:5000) at 4℃ overnight, and wash 3 times with TBST; incubate secondary antibody (HRP label, 1:10000) at room temperature for 1 h, and wash 3 times with TBST; Finally, add enhanced chemiluminescence working solution, and use a chemiluminescence imaging system to detect the expression of C3 and GAPDH proteins, and calculate the C3 to GAPDH ratio.

[0058] Western blot analysis was performed on ectopic endometrial tissue and its matched eutopic endometrial tissue from the same patient, as well as eutopic endometrial tissue from the control group. The results are as follows: Figure 3 As shown, the expression level of C3 in ectopic endometrial tissue was significantly higher than that in eutopic endometrial tissue of EM patients and eutopic endometrial tissue of control patients.

[0059] Example 2: Abnormal expression of C3 and C3aR in an EM mouse model

[0060] EM mouse model construction: An EM mouse model was constructed by intraperitoneal injection of endometrial fragments. Three days before modeling, donor and recipient mice were subcutaneously injected with 3 μg of estrogen to standardize their estrous cycles. On the day of modeling, donor mice were euthanized by CO2 anesthesia and cervical dislocation. The uterus of the donor mice was obtained, and the endometrial fragments were obtained by cutting and resuspending them in PBS. The endometrial fragments were injected into the peritoneal cavity of the recipient mice at a ratio of 2 recipient mice to 1 donor mouse, thus obtaining EM mice.

[0061] The control group consisted of normal, healthy mice. The control group mice were injected intraperitoneally with PBS, while the control group and EM group mice were injected subcutaneously with 3 μg of E2, and the injection was repeated once after 3 days.

[0062] Fourteen days after modeling, mice were anesthetized and euthanized by cervical dislocation. Ascites, uterus and lesions were collected from the mice. Single-cell suspensions were obtained by processing the uterus and lesions. The expression of C3aR in the control group and EM mice was detected by flow cytometry.

[0063] The results are as follows Figure 5 As shown, C3aR expression in ascites cells of EM mice was higher than that in ascites cells of control mice; Figure 6 As shown, C3aR expression in ectopic lesions of EM mice was significantly higher than that in their situ endometrium and in the situ endometrium of control mice; Figure 7 As shown, C3aR is mainly expressed on macrophages, with low expression levels on other immune cells.

[0064] This indicates that, compared with the control group, the expression level of C3aR in the peritoneum and lesion sites of EM mice was significantly increased; and the difference in C3aR expression between the lesion and the in situ endometrium was significantly higher than the difference in C3aR expression in the ascites of the two groups of mice, indicating that there is obvious abnormal complement activation in the EM site, which leads to a significant increase in the expression of C3 and its receptor C3aR in the lesion.

[0065] Example 3: C3aR inhibitors can inhibit lesion growth

[0066] The EM mouse model was established using the method described in Example 2. Seven days after modeling, the EM mice were divided into two groups: a C3aR inhibitor administration group and a no-administration group. Mice in the C3aR inhibitor administration group were intraperitoneally injected with the C3aR inhibitor SB290157, which was dissolved using dimethyl sulfoxide (DMSO) in PBS containing 5% DMSO. Mice in the no-administration group were injected with PBS containing 5% DMSO. Both groups were administered the C3aR inhibitor continuously. Eighteen days after modeling, the mice were sacrificed, and the size of the lesions in both groups was observed.

[0067] The results are as follows Figure 8 As shown, administration of the C3aR inhibitor SB290157 effectively inhibited lesion growth, and the weight and volume of EM lesions were reduced, indicating that administration of C3aR inhibitors can effectively treat EM disease.

[0068] Example 4: C3aR inhibitor administration improves the immunosuppressive microenvironment

[0069] Ascites, uterus, and lesions were collected from mice in the C3aR inhibitor-treated and non-treated groups in Example 3. Flow cytometry analysis was performed to assess macrophage polarization, the number and function of CD8+ T cells, etc.

[0070] like Figure 9 As shown, in the ascites of EM mice, CD206 expression was decreased and iNOS expression was increased in peritoneal macrophages in the C3aR inhibitor administration group. This indicates that after C3aR inhibitor administration, mouse peritoneal macrophages were polarized towards M1, that is, the expression of the pro-inflammatory macrophage M1 marker - inducible nitric oxide synthase (iNOS) increased, and the expression of the anti-inflammatory macrophage M2 marker CD206 decreased.

[0071] like Figure 10 As shown, in ectopic lesions of EM mice, CD206 expression was decreased and iNOS expression was increased in macrophages of the lesions treated with C3aR inhibitors, indicating that the expression trends of CD206 and iNOS in macrophages in ectopic lesion tissues were consistent with those in ascites.

[0072] like Figure 11 As shown, the number of CD8-positive T cells in the ectopic lesions of EM mice in the C3aR inhibitor-treated group was significantly higher than that in the untreated group.

[0073] like Figure 12 As shown, in ectopic lesions of EM mice, the expression of granzyme B in the C3aR inhibitor-treated group was significantly higher than that in the untreated group. Granzyme B (GZMB) can characterize the killing ability of CD8 positive T cells, further indicating that the enhanced killing function of CD8 positive T cells enhances the killing effect on ectopic endometrial stromal cells, thereby inhibiting the progression of lesions.

[0074] Example 5: C3aR inhibitors reduce macrophage-induced endometrial stromal cell migration ability

[0075] The effects of C3a on macrophages were investigated in vitro. The experiment consisted of three groups: a control group (untreated macrophages), a macrophage pretreatment group (macrophages pretreated with C3a), and a macrophage pretreatment group (macrophages pretreated with a combination of C3a and a C3aR inhibitor). Pretreatment lasted 24 hours. Stimulated macrophages were then co-cultured with mouse endometrial stromal cells (MESCs). MESCs were located in the upper chamber of the endothelial cell line, and the treated macrophages were located in the lower chamber. Co-culture continued for 24 hours, and the migration ability of the MESCs was assessed.

[0076] like Figure 13 As shown, compared with untreated macrophages in the control group, C3a-pretreated macrophages enhanced MESC migration ability; compared with C3a-pretreated macrophages, the MESC migration ability was reduced in the group of macrophages pretreated with C3a combined with C3aR inhibitors. This indicates that C3a-pretreated macrophages can enhance MESC migration ability, and this phenomenon was reversed after the addition of C3aR inhibitors. The reduced migration ability of endometrial stromal cells can effectively reduce endometrial adhesion and invasion outside the uterus, and decrease the migration of ectopic endometrial stromal cells from existing lesions to the surrounding area, thereby inhibiting the spread of lesions.

[0077] Example 6: C3, C3a, and C3aR used to diagnose emphysema.

[0078] In Example 1, immunofluorescence and Western blot analysis of ectopic tissues from EM patients and in situ tissues from non-EM patients revealed a significant increase in C3 expression in ectopic lesions of EM patients. In Example 2, the expression of C3 and C3aR was significantly increased in ectopic tissues of EM mice. Given that C3a is a cleavage product of complement C3 and C3aR is a receptor for C3a, and the significant increase in the expression of C3 and C3aR in the ectopic tissues of EM patients indicates increased C3a expression in the ectopic tissues of EM patients. Therefore, EM disease can be diagnosed by detecting the content of any one of C3, C3a, and C3aR alone, or by detecting the content of any two or three of C3, C3a, and C3aR.

[0079] Specifically, EM disease is diagnosed using antibodies targeting C3 and / or C3a and / or C3aR, PCR, qPCR primers, and FISH nucleic acid probes.

[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. The use of C3 and / or C3a and / or C3aR as targets in the preparation of diagnostic and / or therapeutic agents for endometriosis-related diseases.

2. Application of C3 and / or C3a and / or C3aR targets as diagnostic biomarkers in the preparation of reagents or kits for diagnosing endometriosis.

3. The application according to claim 2, characterized in that, The term "endometriosis" includes at least one of the following: uterosacral ligament type endometriosis, peritoneal type endometriosis, rectovaginal type endometriosis, deep infiltrative type endometriosis, ovarian type endometriosis, abdominal wall incision type endometriosis, perineal incision type endometriosis, pulmonary type endometriosis, and nasal cavity type endometriosis.

4. Application of reagents for detecting C3 and / or C3a and / or C3aR in the preparation of kits for diagnosing diseases caused by endometriosis.

5. The application according to claim 4, characterized in that, The reagents for detecting C3 and / or C3a and / or C3aR include at least one of PCR primers, qPCR primers, FISH nucleic acid probes, antibodies, and binding peptides.

6. The use of inhibitors of C3 and / or C3a and / or C3aR in the preparation of drugs for the treatment of endometriosis.

7. The application according to claim 6, characterized in that, The inhibitor is one of gRNA, dsRNA, siRNA, shRNA, or microRNA that targets the DNA and / or RNA of C3 and / or C3aR to inhibit gene transcription and protein expression; or, the inhibitor is a targeted cell that targets the DNA sequence of C3aR, through in vitro gene modification and amplification of autologous cells and their reinfusion.

8. The application according to claim 6, characterized in that, The inhibitor is a biological or chemical agent that targets the protein sequence of C3 and / or C3a and / or C3aR and has an inhibitory or antagonistic effect on C3 and / or C3a and / or C3aR; the biological or chemical agent includes at least one of antibodies, small molecule peptide compounds, and protein degradation-targeting chimeras.

9. The application according to claim 8, characterized in that, The inhibitor is SB290157.

10. A diagnostic kit for endometriosis, characterized in that, Including C3 and / or C3a and / or C3aR targets as diagnostic biomarkers.

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