CN116492462B - Application of PAD4 inhibitor in preventing and treating immune rejection after cornea transplantation - Google Patents
Application of PAD4 inhibitor in preventing and treating immune rejection after cornea transplantation Download PDFInfo
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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Abstract
The invention provides application of PAD4 inhibitor in preventing and treating immune rejection after cornea transplantation, belonging to the technical field of ophthalmic disease medicines. The invention provides an application of a preparation for inhibiting NETs formation in preparing medicines for delaying and/or inhibiting immune rejection after cornea transplantation, wherein PAD4 inhibitor is innovatively applied to preventing and treating the immune rejection after cornea transplantation, for example, BMS-P5 with a certain dosage is injected under conjunctiva, so that the immune rejection of cornea transplantation can be effectively inhibited, the survival time of cornea implants can be prolonged, the preparation can be used for treating the immune rejection after cornea transplantation, and good effects can be obtained.
Description
Technical Field
The invention belongs to the technical field of ophthalmic disease medicines, and particularly relates to application of a PAD4 inhibitor in preventing and treating immune rejection after cornea transplantation.
Background
Cornea blindness is a second approximate blindness eye disease in China, at present, about 400 ten thousand of cornea blindness patients in China can be recovered through cornea transplantation operation, but immune rejection reaction after cornea transplantation operation is caused by transplantation lossThe most significant cause of failure. Immune rejection of cornea transplantations is mainly composed of CD4 + T cell mediated, its process mainly includes: antigen presenting cells (Antigenpresenting Cell, APC) recognize corneal antigens, transmit signals to cervical lymph nodes, proliferate T cells, activate aggressive helper T cells (Th), and then attack corneal implants via vascular or lymphatic migration to cause immune rejection. At present, medicaments such as glucocorticoid, cyclosporin A eye drops, tacrolimus eye drops and the like are mainly applied clinically to prevent and treat cornea transplantation immune rejection reaction, but the anti-rejection effect is not ideal. The main reason is that the mechanism of the antagonism rejection reaction of the medicine is mainly targeted to inhibit lymphocyte proliferation, namely, the mechanism plays a role in the middle link of the immune rejection reaction, but does not interfere with the source of the initial link of immune signal recognition. Therefore, how to perform effective intervention strategies in the initial stage of the initiation of the immune rejection reaction, thereby blocking the subsequent immune response, and having important clinical application value for preventing and treating the cornea transplantation immune rejection reaction and improving the prognosis of the cornea blind patient.
The initiation of immune rejection is initiated by antigen signaling and activation by APC followed by activation of primary CD4 either directly or indirectly + T cells. While it has been recognized that APC plays a key role in mediating graft immune rejection, the specific molecular mechanism of APC activation during immune rejection is not yet understood. Studies have shown that neutrophil extracellular traps (neutrophil extracellulartraps, nes) promote APC activation, initiate an immune response in the body, and thus cause tissue damage after organ transplantation. NETs are composed mainly of extracellular DNA and granular antimicrobial proteins, including citrullinated histones, elastase, myeloperoxidase, and the like. Protein arginine deaminase 4 (PAD 4) -driven histone citrullination is required in the process of forming the nes, and studies show that inhibiting PAD4 activity can block the formation of the nes, regulate innate immune responses and alleviate vascular injury in atherosclerosis mice. However, the effectiveness of PAD4 inhibitors for treating immune rejection following corneal transplantation has not been reported.
Disclosure of Invention
The invention aims to provide an application of PAD4 inhibitor in preventing and treating immune rejection after cornea transplantation, and the PAD4 inhibitor is used for preparing a medicament for inhibiting the immune rejection after cornea transplantation, so that the effect is good.
The invention provides an application of a preparation for inhibiting the formation of Neutrophil Extracellular Traps (NETs) in preparing a medicament for delaying and/or inhibiting immune rejection after cornea transplantation.
Preferably, the agent that inhibits neutrophil extracellular trap formation comprises an arginine deaminase 4 (PAD 4) inhibitor.
Preferably, the agent that inhibits neutrophil extracellular trap formation comprises BMS-P5.
Preferably, the pharmacologically effective concentration of BMS-P5 is 4-60 mg/ml.
Preferably, the pharmacologically effective concentration of BMS-P5 is 5mg/mL.
The invention also provides application of BMS-P5 in preparing anti-inflammatory drugs for inhibiting I-type IFN related channels.
The invention also provides a medicament for inhibiting cornea transplant immune rejection reaction, which comprises BMS-P5 with pharmacological effective concentration.
Preferably, the medicament is in the form of injection.
The beneficial effects are that: the invention provides an application of a preparation for inhibiting NETs formation in preparing medicines for delaying and/or inhibiting immune rejection after cornea transplantation, wherein PAD4 inhibitor is innovatively applied to prevention and treatment of immune rejection after cornea transplantation, PAD4 inhibitor BMS-P5 is selected in examples to prevent and treat immune rejection after cornea transplantation, NETs expression is increased in cornea implants for generating immune rejection, and after allogeneic mice cornea transplantation, BMS-P5 (such as 5 mg/mL) is injected under conjunctiva at a certain dose, so that the immune rejection after cornea transplantation can be effectively inhibited, the survival time of the cornea implants can be prolonged, and the preparation can be used for treatment of immune rejection after cornea transplantation, and good effects can be obtained.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing immunoblotting and immunofluorescence staining results of a mouse corneal implant without rejection in normal, syngeneic, and allogenic corneal transplants;
FIG. 2 is a graph showing the effect of subconjunctival injection of 5mg/mL of BMS-P5, a drug that inhibits NETs formation, on immune rejection following a corneal graft.
Detailed Description
The invention provides an application of a preparation for inhibiting the formation of Neutrophil Extracellular Traps (NETs) in preparing a medicament for delaying and/or inhibiting immune rejection after cornea transplantation.
Formulations of the invention that inhibit the formation of NETs include an arginine deaminase 4 (PAD 4) inhibitor, and more preferably BMS-P5. The pharmacologically effective concentration of BMS-P5 of the present invention is preferably 4 to 60mg/mL, more preferably 5mg/mL. BMS-P5 according to the invention is preferably derived from Glpbio.
The invention also provides application of BMS-P5 in preparing anti-inflammatory drugs for inhibiting I-type IFN related channels.
In the embodiment of the invention, the intervention treatment of PAD4 inhibitor BMS-P5 is utilized to obviously inhibit the immune rejection of cornea transplantation, and the expression of a leukocyte surface active marker CD45 is obviously reduced after the BMS-P5 is injected, which proves that the subconjunctival injection of BMS-P5 of an allogeneic cornea transplantation rejection mouse can reduce the quantity of leukocytes in cornea implants and lighten the inflammatory reaction of cornea transplantation immune rejection; and the expression level of genes such as IFN-gamma, IL-1 beta, CXCL-10 and the like of the I-type IFN related inflammatory factors is reduced, namely BMS-P5 plays an anti-inflammatory role by inhibiting the I-type IFN related pathway.
The invention also provides a medicament for inhibiting cornea transplant immune rejection reaction, which comprises BMS-P5 with pharmacological effective concentration.
The medicament of the invention is preferably injection, and the pharmacological effective concentration is preferably 5mg/mL.
For further explanation of the present invention, the application of the PAD4 inhibitor provided in the present invention in preventing and treating immune rejection after cornea implantation is described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
EXAMPLE 1 increased expression of NETs in mouse corneal grafts following corneal transplantation with immune rejection
1. Male BALB/c mice of 8-10 weeks old were divided into the following 3 groups using a completely randomized block design:
A. mice not subjected to cornea transplantation served as a normal control group;
B. syngeneic allogeneic cornea transplant groups (donor is same week-old BALB/c mice);
C. allogeneic cornea transplant groups (donor is C57BL/6 mice of the same week), 6-8 of each group.
The penetrating cornea transplant model is established as follows: the mice are anesthetized by intraperitoneal injection of 0.6% pentobarbital sodium, all mice are subjected to right eye operation, conjunctival sac is flushed with sterile physiological saline before operation, and the compound topiramate eye drops are fully mydriatic, and the whole operation is carried out under a microscope. Taking the central cornea of a donor mouse as a implant by using a 2.5mm trephine, removing the central cornea of a recipient mouse by 2mm to prepare an implant bed, and intermittently suturing 8 needles by using 11-0 nylon suture lines to fix the implant on the implant bed. After the operation, air is injected into the anterior chamber to form the anterior chamber, ofloxacin eye ointment is coated in conjunctival sac to prevent infection, and the eyelid of the recipient mouse is sutured by using a 10-0 nylon suture. The corneal suture was removed on postoperative day 7. The slit lamp microscope was observed 3 times a week, and mice with complications such as cataract, iris adhesion, anterior chamber hemorrhage, infection, etc. were removed and supplemented with corresponding animals.
2. On the 19 th day after surgery, mice were sacrificed, eyeballs of group a and group C mice were taken, 3 samples each, and OCT gel was embedded and frozen into sections with a section thickness of 7 μm. The OCT gel was washed off with PBS, blotted around the specimen with a histochemical pen, the specimen was fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X, and blocked with 5% BSA for 1h at room temperature. The slides were spun dry and incubated for 4H at room temperature with rabbit anti-mouse H3cit antibody (1:200) and rat anti-mouse Ly6G antibody (1:100). The primary antibody was washed off with PBS and Alexa Fluor 594 donkey anti-rabbit antibody (1:1000) and Alexa Fluor 488 sheep anti-rat antibody (1:500) were co-incubated at room temperature for 1h. After washing off the antibodies, nuclei were stained with DAPI solution and observed under a positive inverted microscope (a in fig. 1). It was found that H3cit expression was significantly increased in the stromal and endothelial layers of the C-group implant cornea. H3cit is a marker for NETs formation, and an increase in expression level indicates an increase in NETs expression, and the above results indicate an increase in NETs formation in the corneal implant when the corneal graft rejection reaction occurs.
3. On the 19 th day after operation, mice were sacrificed by cervical removal, 3 mice per group were each obtained, the whole cornea was cut off along the limbus and placed in a 1.5ml centrifuge tube, pre-chilled RIPA lysate and milling beads were added, PMSF protease inhibitor was added at 1% concentration, sample proteins were extracted by tissue milling and ultrasound, and the proteins were denatured by a metal bath at 95 ℃ for 10 min. Proteins were separated by SDS-PAGE gel electrophoresis, transferred to PVDF membrane, blocked with 5% BSA at room temperature for 2H, and incubated overnight at 4℃with the addition of rabbit anti-H3 cit antibody (1:1000) and rabbit anti- β -actin antibody (1:1000). The following day was incubated with goat anti-rabbit IgG-HRP secondary antibody at room temperature for 2h after TBST washing, developed with ECL luminescence kit, and exposed with Bio-Rad rapid luminescence photographic apparatus (FIG. 1B). The results show that the protein expression level of the group C is obviously increased compared with that of the group A and the group B by taking the beta-actin as an internal reference. Citrullinated histones are markers of NETs formation, and increased expression levels indicate increased NETs expression, thus the results indicate increased NETs expression in implants following corneal transplantation where immune rejection occurs.
EXAMPLE 2PAD4 inhibitor BMS-P5 intervention treatment significantly inhibited corneal graft immune rejection
1. Using a completely randomized block design, 8-10 week old male BALB/c mice were randomly drawn into the following 3 groups:
A. mice not subjected to cornea transplantation served as a normal control group;
B. allogeneic cornea transplant groups;
C. allogeneic cornea transplant + BMS-P5 groups of 6-8 animals each.
A penetrating cornea implant model was established as described above. The drug was injected subconjunctival at 4 days post-surgery, with group B PBS solution, group C BMS-P5 at 5mg/kg and frequency of 1/3 d for 4 total injections. After operation, observation and photographing are carried out under a slit lamp every 1 day (figure 2), three indexes including transparency, edema degree and neovascularization of the cornea implant are scored by referring to a Larkin scoring method, the total observation time is 30 days, the occurrence time of each group of rejection reactions is recorded, and a cornea implant rejection survival curve is drawn.
As a result, on the 14 th day after the operation, the allograft group cornea implant gradually appears obvious edema, turbidity, peeping of the pupil and rejection reaction, while the BMS-P5 injection group cornea implant still keeps transparent. The time to rejection of the corneal implants was significantly prolonged in the BMS-P5 injected group compared to the allograft group (FIG. 2). Because PAD4 plays a key role in the NET formation process, the PAD4 inhibitor BMS-P5 can effectively inhibit NETs formation, so that the survival rate of the cornea implant is improved, and the immune rejection of cornea transplantation is inhibited.
2. On day 19 post-surgery, 3 eyes were taken for each group, the remaining mice were continuously observed, and OCT gel was embedded and frozen into sections. The OCT gel was washed off with PBS, blotted around the specimen with a histochemical pen, the specimen was fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X, and blocked with 5% BSA for 1h at room temperature. The slides were spun dry and the rabbit anti-CD 45 antibody incubated overnight at 4 ℃. The next day the primary antibody was washed off, the nuclei stained with DAPI solution and observed under a positive inverted microscope. As a result, the expression of the C-group leukocyte surface active marker CD45 was found to be significantly reduced as compared with the B-group, indicating that subconjunctival injection of BMS-P5 in allogeneic cornea transplant rejection mice can reduce the number of leukocytes in cornea implants and reduce the inflammatory response of cornea transplant immune rejection.
3. On the 19 th day after operation, 3 mice are taken from each group, the whole cornea is cut along the limbus, the total RNA of each group of cornea tissues is extracted by a Trizol method, the concentration of the RNA is measured on a Nanodrop, the mRNA is reversely transcribed into cDNA by a reverse transcription kit, a reaction solution is prepared by referring to the instruction book of the PCR kit, and each group of cDNA is added, and fluorescent quantitative PCR detection is carried out by taking GAPDH as an internal reference.
The primer sequences were as follows:
GAPDH upstream primer (SEQ ID No. 1): 5'-GCCACCCAGAAGACTGTGGAT-3' the number of the individual pieces of the plastic,
downstream primer (SEQ ID NO. 2): 5'-GGAAGGCCATGCCAGTGA-3';
IFN-gamma upstream primer (SEQ ID NO. 3): 5'-TCTTGGGTTCTTACGGCTGTTAC-3' the number of the individual pieces of the plastic,
downstream primer (SEQ ID NO. 4): 5'-TGTCACTCTCCTCTTTCCAATTCC-3';
IL-1. Beta. Upstream primer (SEQ ID NO. 5): 5'-CTTTCCCGTGGACCTTCCA-3' the number of the individual pieces of the plastic,
downstream primer (SEQ ID NO. 6): 5'-CTCGGAGCCTGTAGTGCAGTT-3';
CXCL-10 upstream primer (SEQ ID NO. 7): 5'-GGTCCGCTGCAACTGCAT-3' the number of the individual pieces of the plastic,
downstream primer (SEQ ID NO. 8): 5'-GGATTCAGACATCTCTGCTCATCA-3'.
By 2 —△△CT The method is used for relative quantitative standardization of data. The results show that the expression level of the I-type IFN related inflammatory factors IFN-gamma, IL-1 beta, CXCL-10 and the like is reduced. The results show that the expression level of inflammatory factor genes of the cornea implant in the BMS-P5 injection group is obviously lower than that in the allograft group, and the BMS-P5 plays an anti-inflammatory role by inhibiting the I-type IFN related pathway.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (3)
- Use of bms-P5 in the preparation of a medicament for delaying and/or inhibiting immune rejection following a corneal transplantation procedure.
- 2. The use according to claim 1, wherein the pharmacologically effective concentration of BMS-P5 is 4-60 mg/ml.
- 3. The use according to claim 2, characterized in that the pharmacologically effective concentration of BMS-P5 is 5mg/mL.
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| CN113633642A (en) * | 2021-08-26 | 2021-11-12 | 山东第一医科大学附属青岛眼科医院(山东省眼科研究所、青岛眼科医院) | Application of ABT-263 in preparation of medicine for inhibiting corneal transplantation immune rejection |
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