阅读说明：本技术 Nl101在制备治疗急性、慢性排斥反应药物中的应用 (Application of NL101 in preparation of medicine for treating acute and chronic rejection ) 是由 沈佳 葛求富 刘帅辉 郭陆英 严芃芃 黄洪锋 于 2021-03-24 设计创作，主要内容包括：本发明公开了NL101在制备治疗急性、慢性排斥反应药物中的应用。本发明通过脾脏致敏诱导的急性肾移植抗体介导的排斥反应模型,研究NL101对急性排斥反应的抑制作用,实验表明腹腔注射NL101,能显著减少异型肾脏移植小鼠外周血、脾脏细胞的B细胞和浆细胞群落比例,减轻急性排斥反应。同样地,NL101对慢性排斥反应具有抑制作用,能够显著降低异型肾脏移植小鼠的死亡率。(The invention discloses an application of NL101 in preparing a medicine for treating acute and chronic rejection. According to the invention, through an acute kidney transplantation antibody-mediated rejection model induced by spleen sensitization, the inhibition effect of NL101 on acute rejection is researched, and experiments show that the injection of NL101 into the abdominal cavity can obviously reduce the proportion of B cell and plasma cell populations of peripheral blood and spleen cells of a heterotypic kidney transplantation mouse and alleviate the acute rejection. Similarly, NL101 has an inhibitory effect on chronic rejection and can significantly reduce the mortality in heterotypic kidney transplant mice.)

Use of NL101 for the manufacture of a medicament for the treatment of acute or chronic rejection.

2. Use of NL101 according to claim 1 for the manufacture of a medicament for the treatment of acute or chronic rejection, wherein the medicament comprises at least NL 101.

Use of NL101 for the manufacture of a medicament for the treatment of acute or chronic ABMR.

Application of NL101 in preparation of medicine for treating spleen sensitization-induced acute kidney transplantation antibody-mediated rejection.

Technical Field

The invention relates to the technical field of medicines, in particular to application of NL101 in preparation of medicines for treating acute and chronic rejection.

Background

Antibody-mediated rejection (ABMR) is a significant cause of graft failure. ABMR occurs mainly from mismatch of Human Leukocyte Antigens (HLA) between donor and recipient, and Antigen Presenting Cells (APCs) process HLA antigens exposed in transplanted organs into antigenic peptides and present them to mature B cells, develop activated B cells at Germinal Centers (GC) with the help of helper T cells, and finally differentiate into plasma cells secreting antibodies (DSA) against donor specificity; in the case of ABMR relapse, the proliferation of plasma cells follows another more convenient approach, i.e., memory B cells are activated and proliferated directly under the coordination of helper T cells, and DSA-secreting plasma cells are produced in large quantities in a short time. After the secreted DSA is combined with graft endothelial cells, on one hand, immune cells expressing Fc gamma R can be independently recruited, on the other hand, complement activation is triggered, and a Membrane Attack Complex (MAC) is formed at the vascular cavity side of the endothelial cells, so that endothelial stress and inflammatory cell recruitment signals are further enhanced.

HLA differentiation is due to genes, complex sites, and incomplete clearance or blockage under state-of-the-art conditions, and therefore, how to reduce and block DSA-producing plasma cell differentiation and proliferation is a key point for ABMR therapy in ABMR injury.

The main goals of current ABMR therapy include:

1) reducing or eliminating new DSA generation, 2) eliminating existing DSA in circulation, 3) blocking the effect of circulating DSA on transplants, only CD20 monoclonal antibody to eliminate B cells, plasmapheresis and gamma globulin clearance (blocking) antibodies, hormone shock to reduce inflammation or combination regimens, although these treatments may improve short-term prognosis, have also been shown to fail to improve long-term prognosis (Am J Transplant 2009; 1099-107.Am J Transplant 2011; 2405-13.N Engl J Med 2018; 379:1150-60). The lack of organ and site targeting is a commonality of these treatments.

NL101 (i.e., EDO-S101), the structure is shown below,

NL101 has the formula C₁₉H₂₈Cl₂N₄O₂A molecular weight of 415.36, white to pale yellow crystalline powder; no bad smell. Is slightly soluble in dimethyl sulfoxide, slightly soluble in methanol or N, N-dimethylformamide, insoluble or almost insoluble in water, and soluble in acetic acid.

NL-101 is a drug that acts on HDAC molecules and DNA molecules in tumor cells to produce a definite anti-tumor effect. HDAC and DNA have been clinically proven as effective anti-malignant tumor targets, the former being a modern molecular targeting target and the latter being a traditional cytotoxic target. Inhibition of HDAC activity after NL-101 entry into tumor cells: on the one hand, tightly bound chromatin (DNA) becomes loose, making it more susceptible to attack by the nitrogen mustard group in the NL-101 molecule, causing massive DNA damage, inducing apoptosis and cycle arrest, and on the other hand, inhibiting DNA damage repair by direct or indirect means, preventing cell survival and development of drug resistance. There is currently no report of the use of NL-101 for the treatment of acute and chronic rejection.

Disclosure of Invention

In view of the above, the present invention provides for the use of NL101 for the manufacture of a medicament for the treatment of acute and chronic rejection in order to overcome the disadvantages of the prior art.

In order to achieve the purpose, the invention provides the following technical scheme:

application of NL101 in preparing medicine for treating acute and chronic rejection.

Further, the acute and chronic rejection drug at least contains NL 101.

Application of NL101 in preparing medicine for treating acute and chronic ABMR is disclosed.

Application of NL101 in preparation of medicine for treating spleen sensitization-induced acute kidney transplantation antibody-mediated rejection.

The invention has the beneficial effects that:

according to the invention, through an acute kidney transplantation antibody-mediated rejection model induced by spleen sensitization, the inhibition effect of NL101 on acute rejection is researched, and experiments show that the injection of NL101 into the abdominal cavity can obviously reduce the proportion of B cell and plasma cell populations of peripheral blood and spleen cells of a heterotypic kidney transplantation mouse and alleviate the acute rejection. Similarly, NL101 has an inhibitory effect on chronic rejection and can significantly reduce the mortality in heterotypic kidney transplant mice.

Drawings

FIG. 1 is a schematic diagram and a statistical diagram of the detection results of B cells and plasma cells in peripheral blood and spleen cells of mice in an antigen sensitized group, a homo-kidney transplantation group and a hetero-kidney transplantation induced acute ABMR group, an ABMR + tacrolimus treatment group and an ABMR + NL101 treatment group.

FIG. 2 is a statistical chart of the detection results of the serum IgG and IgM abundances of mice in the antigen sensitized group, the homo-kidney transplantation group, and the hetero-kidney transplantation induced acute ABMR group, the ABMR + tacrolimus treatment group, and the ABMR + NL101 treatment group according to the present invention.

FIG. 3 is a C4d fluorescence immunity diagram in transplanted kidneys of antigen sensitized group, homo-kidney transplanted group, hetero-kidney transplanted group resulting in acute ABMR group, ABMR + tacrolimus treatment group and ABMR + NL101 treatment group according to the present invention.

FIG. 4 is a diagram showing the proportional flow detection and statistics of lymphocytes in transplanted kidneys of the antigen-sensitized group, the homo-kidney transplantation group and the hetero-kidney transplantation induced acute ABMR group, the ABMR + tacrolimus treatment group and the ABMR + NL101 treatment group of the invention.

FIG. 5 is a statistical graph showing the levels of IL-6, IL-10, MCP-1, IFN-. gamma., TNF and IL-12p70 in the serum and transplanted kidney of the antigen-sensitized group, the homo-kidney transplantation group, and the hetero-kidney transplantation resulted in the acute ABMR group, the ABMR + tacrolimus treatment, and the ABMR + NL101 treatment group according to the present invention.

FIG. 6 is a graph of mortality results for each group of chronic ABMR.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments provided in the present application without any inventive step are within the scope of protection of the present application.

Reference in the specification to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the specification. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of ordinary skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments without conflict.

Unless defined otherwise, technical or scientific terms referred to herein shall have the ordinary meaning as understood by those of ordinary skill in the art to which this application belongs. Reference to "a," "an," "the," and similar words throughout this application are not to be construed as limiting in number, and may refer to the singular or the plural. The present application is directed to the use of the terms "including," "comprising," "having," and any variations thereof, which are intended to cover non-exclusive inclusions; reference to "connected," "coupled," and the like in this application is not intended to be limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. Reference herein to "a plurality" means greater than or equal to two. "and/or" describes an association relationship of associated objects, meaning that three relationships may exist, for example, "A and/or B" may mean: a exists alone, A and B exist simultaneously, and B exists alone. Reference herein to the terms "first," "second," "third," and the like, are merely to distinguish similar objects and do not denote a particular ordering for the objects.

Reagents, drugs, instruments, etc. used in the following examples are commercially available.

Example 1

1. Animal grouping and surgery

Male C57BL/6 and Balb/C mice, which are 10 weeks old, both grow freely before model preparation in sterile environment with temperature of 23 + -2 deg.C and humidity of 55 + -10%, and the body weight is about 25 g. In the operation period, pentobarbital physiological saline solution with the mass fraction of 1 percent is used for carrying out intraperitoneal injection on an anesthetized mouse (80mg/kg mouse), the operation treatment is divided into two stages of antigen sensitization and graft rejection, and the specific grouping and implementation steps are as follows:

(1) antigen-sensitized group (antigen-induced group, simply referred to as Blank group): spleen of Balb/c male mouse is taken and blunt separated to prepare the cell with the concentration of 10⁷Spleen single cell physiological saline suspension per ml for standby. Freshly prepared single cell suspensions of the above spleens (0.1 ml/mouse) were injected into C57BL/6 male mice via tail vein and were not transplanted after sensitization (n-4, i.e., 4C 57BL/6 male mice were not transplanted), and their kidneys were used as a blank control.

Other antigen-sensitized C57BL/6 male mice were used as recipients and are shown in the following groups. The donor C57BL/6 mice described in the following cohorts were not sensitized with Balb/C mouse antigen.

(2) Homo-renal transplantation group (Iso group for short): taking 4C 57BL/6 mice sensitized by Balb/C mouse antigen as a receptor, taking the kidney of a donor C57BL/6 mouse, transplanting the kidney to a receptor C57BL/6 mouse, and keeping the original kidney of the receptor mouse;

(3) heterotypic kidney transplantation leads to the acute ABMR group (allogenic kidney transplantation induced acute AMBR group, abbreviated ABMR group): taking 4 Balb/C mouse antigens sensitized C57BL/6 mice as a receptor, taking Balb/C mice as a kidney donor, transplanting the Balb/C mice to the receptor C57BL/6 mice, and keeping the protonephron of the receptor mice; because the receptor C57BL/6 mouse is sensitized by Balb/C mouse antigen 2 weeks before to generate antibody, acute antibody-mediated rejection reaction can be generated when the receptor C57BL/6 mouse contacts Balb/C mouse-derived transplant for the second time;

(4) ABMR + Tacrolimus treatment group (ABMR + Tacrolimus group, abbreviated ABMR + Tac group): taking 4 Balb/C mouse antigens sensitized C57BL/6 mice as a receptor, taking Balb/C mice as a kidney donor, transplanting the Balb/C mice to the receptor C57BL/6 mice, keeping the original kidney of the receptor mice, and performing intraperitoneal injection treatment on the receptor mice with Tac solution (namely tacrolimus solution or FK506 solution) every day after operation; the preparation and injection amounts of the Tac solution are detailed below: and (5) treating the medicine.

(5) ABMR + NL101 treatment group (ABMR + NL101 group, abbreviated ABMR + NL101 group): taking 4C 57BL/6 mice sensitized by Balb/C mouse antigen as a receptor, taking Balb/C mice as a kidney donor, transplanting the Balb/C mice to the receptor C57BL/6 mice, keeping the proto-kidney of the receptor mice, and treating the receptor C57BL/6 mice with NL101 solution 24 hours before transplanting; the preparation and injection quantities of the NL101 solution are detailed below: and (5) treating the medicine.

2. Drug treatment

The preparation and injection dosage of the Tac solution are as follows:

tacrolimus (FK506) powder was dissolved in DMSO corn oil solvent with a volume fraction of 5% to prepare a stock solution Y with a concentration of 15mg/ml, stored at-80 ℃, diluted with physiological saline to a tacrolimus solution with a concentration of 0.125mg/ml before use, and subject mice were intraperitoneally injected with the FK506 solution (1mg FK506/kg mice) 30 minutes before replating transplanted kidneys, and the dose was maintained for daily administration until 5 days after surgery, and mice were sacrificed and sampled.

The NL101 solutions were prepared and injected in the following amounts:

NL101 was dissolved in DMSO to prepare 10mg/ml stock solution X, stored at 4 ℃ protected from light, prepared into NL101 solution with a concentration of 2mg/ml with physiological saline before use, and administered to recipient C57BL/6 mice intraperitoneally 24h before transplantation (30mg NL101 solution/kg mice).

The antigen sensitized group and the homotype kidney transplantation group were administered the same volume of physiological saline for intraperitoneal injection, and were not administered other drugs.

After 5 days after transplantation, injecting 1% pentobarbital normal saline solution (80mg/kg) into abdominal cavity to anaesthetize mouse, collecting whole blood in anticoagulation tube by eyeball-picking blood-taking method; all mice were injected intraperitoneally with 5% by mass pentobarbital saline solution (300mg pentobarbital/kg mice), anesthetized, euthanized, and spleen and kidney tissues were taken for subsequent detection.

3. Detection of new plasma cells

Mixing 0.2ml of whole mouse blood with 2ml of erythrocyte lysate, incubating for 15min at normal temperature in a dark place, centrifuging for 400g, and extracting peripheral blood cells (PBMC) for 5 min; the spleen is pressed by a grinding head through a 40 mu m filter screen and is subjected to blunt separation to form spleen single cell suspension; the PBMC and spleen single cells were resuspended in BSA/PBS solution (1% BSA) to 10 per ml⁸The cell suspensions were prepared by adding 50. mu.l of cell suspension to an equal volume of freshly prepared flow antibody solution (in 50. mu.l volume: for example, CD45 BV786, CD3 FITC, B220 PE-Cy7, CD19 APC-Cy7, CD138 BV421, CD27 PE 1. mu.l + 44. mu.l each of flow cytostaining solution, all reagents purchased from BD Pharmingen), protected from light, incubated on ice for 15min, 2ml of flow cytostaining solution was added, centrifuged for 450g 5min, the supernatant discarded, resuspended with 200. mu.l of PBS (0.01M, pH 7.4), and subjected to flow assay using a Cytoflex LX flow cytometer to measure mainly the ratio of B cells (CD45+ CD 3-B220 + CD19+) to the total population of lymphocytes, and where the plasma cells (CD138+ CD27-) population of lymphocytes, for assessing the effect of NL101 on the level of B and plasma cell activation in ABMR.

4. Serum DSA abundance detection

Mouse whole blood was centrifuged at 3000g for 30min to obtain serum, which was then subjected to IgG and IgM abundance flow cytometry to measure neonatal donor-specific antibody (donor specific antibody) levels for evaluation of NL101 effect on DSA secretion by plasma cells in ABMR reactions.

C4d immunohistochemical detection

Transplanted kidney tissues were fixed with mass fraction 4% paraformaldehyde for 2 hours, dehydrated and sedimented in 20% and 30% gradient sucrose PBS solution (0.01M, pH 7.4), cryosectioned (section thickness 20 μ M), examined for C4d immunohistochemistry on sectioned tissues, and fluorescence photographed for evaluation of the effect of NL101 on complement and endothelial activation levels in ABMR reactions.

6. Graft infiltration inflammatory cell detection

Mice were transplanted with single cell suspensions of kidney tissue, resuspended using BSA/PBS solution (1% BSA), and prepared at 10 per ml⁸For each cell suspension, 50. mu.l of the cell suspension was added to an equal volume of freshly prepared flow antibody solution (50% w/w)μ l volume is for example: CD45 BV786 antibody 1. mu.l + 49. mu.l of flow cytostaining solution, all reagents purchased from BD Pharmingen, protected from light, incubated on ice for 15min, 2ml of flow cytostaining solution added, centrifuged for 450g for 5min, supernatant discarded, resuspended in 200. mu.l PBS (0.01M, pH 7.4), flow-assayed using a Cytoflex LX flow cytometer, and the number of infiltrating lymphocytes (CD45+) in transplanted kidney tissue was determined to assess the effect of NL101 on the level of lymphocyte challenge following activation of the graft endothelium in ABMR reactions.

7. Inflammatory factor level determination

Mouse serum (serum obtained after centrifugation of 3000g 30min of whole mouse blood) and transplanted kidney tissue homogenate were centrifuged for 14,000g 30min, and the supernatant was collected and measured for IL-6, IL-10, MCP-1, IFN-. gamma., TNF, IL-12p70 levels in the samples using the CBA inflammatory factor assay kit (BD, 552364) to assess the effect of NL101 on local and systemic inflammatory levels in ABMR responses.

Example 2

1. Animal grouping and surgery

Male C57BL/6 and Balb/C mice, which are 10 weeks old, both grow freely before model preparation in sterile environment with temperature of 23 + -2 deg.C and humidity of 55 + -10%, and the body weight is about 25 g. In the operation period, pentobarbital physiological saline solution with the mass fraction of 1 percent is used for carrying out intraperitoneal injection on an anesthetized mouse (80mg/kg mouse), the operation treatment is divided into two stages of antigen sensitization and graft rejection, and the specific grouping and implementation steps are as follows:

in each of the following groups, both donor mice and recipient mice were normal mice before kidney transplantation, and were not antigen-sensitized.

(1) Homo-renal transplantation group (Iso group for short): taking 5C 57BL/6 mice as a receptor, taking the kidney of a donor C57BL/6 mouse to transplant to a receptor C57BL/6 mouse, and removing the protokidney of the receptor mouse;

(2) chronic ABMR group caused by heterotypic kidney transplantation (acquired renal AMBR group, abbreviated as "chronic ABMR group"): taking 5C 57BL/6 mice as a receptor, taking Balb/C mice as a kidney donor, transplanting the mice to the receptor C57BL/6 mice, and removing the protonephros of the receptor mice; because the receptor mouse is stimulated by Balb/c mouse antigen for the first time, the antibody has longer generation time and presents a gradual amplification effect, and can generate rejection reaction similar to the mediation of a chronic antibody;

(3) cyclic abrr + Tacrolimus treatment group (cyclic abrr + Tacrolimus group, abbreviated cyclic abrr + Tac): taking 5C 57BL/6 mice as a receptor, taking Balb/C mice as a kidney donor, transplanting the mice to the receptor C57BL/6 mice, and removing the protonephros of the receptor mice; after the operation, the recipient mice are treated by intraperitoneal injection of Tac solution (namely tacrolimus solution or FK506 solution) every day; the preparation and injection dosage of the Tac solution are the same as those in example 1;

(4) cyclic abrr + NL101 treatment group (cyclic abrr + NL101 group, referred to as cyclic abrr + NL101 group): taking 5C 57BL/6 mice as a receptor, taking Balb/C mice as a kidney donor, transplanting the mice to the receptor C57BL/6 mice, and removing the protonephros of the receptor mice; recipient C57BL/6 mouse NL101 solution was given 24 hours prior to transplantation, and the NL101 solution was prepared and injected in the same amount as in example 1.

2. Mouse mortality record

The transplanted mice are independently raised 7 days after operation, so that the influence of fighting on experimental results of the mice is prevented, dead mice are collected every day, anatomical examination is carried out, and the dead mice are brought into the body after surgical operation reasons such as bleeding and urethral obstruction are eliminated.

The result of the detection

1. Detecting results of the number and proportion of newly activated B cells and plasma cells

FIG. 1 is a schematic and statistical chart of the detection results of B cells and plasma cells in peripheral blood and spleen cells in an antigen-sensitized group, a homo-kidney transplantation group, a hetero-kidney transplantation induced acute ABMR group, an ABMR + tacrolimus treatment group, an ABMR + NL101 treatment group, and an ABMR + NL101 treatment group. Peripheral circulating blood cells (shown in FIG. 1A) and spleen cells (shown in FIG. 1B) of mice in antigen-sensitized group (Blank), homo-kidney transplant group (Iso), heterotypic kidney transplant resulting in acute ABMR group (ABMR), ABMR + tacrolimus-treated group (ABMR + Tac), and ABMR + NL 101-treated group (ABMR + NL101) were subjected to flow-type treatmentThe results of the cytometric analysis are shown in FIG. 1; wherein the cell population marks are all the percentage in the upper-order mother population; FIG. 1C is a statistical graph of the percentage of B cells (B cells) and Plasma cells (Plasma cells) in lymphocytes from peripheral circulating blood cells (PBMC) and Spleen cells (Spleen)<0.0001,***P<0.001,and*P <0.05 vs Blank group；^####P<0.0001,^###P<0.001,^##P<0.01,and^#P<0.05 vs Iso group,^ΔΔΔΔP<0.0001,^ΔΔΔP<0.001,^ΔΔP<0.01,and^ΔP<0.05 vs ABMR group；^□□□□P<0.0001 and^□□□P<0.001 vs ABMR+Tac group。

As can be seen in FIG. 1, NL101 significantly reduced the B cell and plasma cell population in the ABMR-responsive spleen. Specifically, in the spleen, there was a significant increase in both homorenal transplant group mouse B cells (B220+ CD19+ group in CD45+ CD 3-group, P <0.01) and plasma cells (CD138+ CD 27-group in B220+ CD19+ group, P <0.05) compared to non-transplant mice (i.e., blank control group).

After the abnormal kidney transplantation results in mouse ABMR, the population ratio of B cells (P <0.0001) and plasma cells (P <0.0001) is obviously improved compared with that of the mouse transplanted to the same type.

Daily tacrolimus administration significantly reduced the proportion of B cell (P <0.001) and plasma cell (P <0.001) populations after rejection of xenogenic kidney transplants; a single injection of NL101 significantly reduced the proportion of B cell (P <0.0001) and plasma cell (P <0.0001) populations induced by ABMR, and was still significantly reduced (P <0.0001) compared to tacrolimus-dosed groups (fig. 1A, C).

In peripheral blood, there was no significant difference between the B cells and plasma cells in the kidney-transplanted mice of the same type as compared with non-transplanted mice. After the abnormal kidney transplantation causes the mouse ABMR, the colony ratio of B cells (P <0.0001) and plasma cells (P <0.0001) is obviously improved compared with that of the abnormal kidney transplantation mouse; daily tacrolimus administration significantly reduced the proportion of B cell (P <0.01) and plasma cell (P <0.01) populations after rejection of xenorenal transplants; treatment with a single injection of NL101 significantly reduced the proportion of B cell (P <0.0001) and plasma cell (P <0.0001) colonies increased by ABMR and still significantly decreased (P <0.001) compared to the tacrolimus-dosed group (fig. 1B, C).

2. Serum DSA level detection result

FIG. 2 is a flow cytometric histogram of IgG and IgM abundance in serum samples from mice in the antigen-sensitized group (Blank), the homo-kidney transplant group (Iso for short), the heterotypic kidney transplant group (ABMR), the ABMR + tacrolimus treated group (ABMR + Tac), and the ABMR + NL101 treated group (ABMR + NL101), where P is represented as the percentage of IgG or IgM positive cells to all cells (% of Parent, ordinate), where<0.001,**P<0.01,and*P<0.05 vs Blank group；^###P<0.001,^##P<0.01,and^#P<0.05 vs Iso group；^ΔΔΔP<0.001,^ΔΔP<0.01,and^ΔP <0.05 vs ABMR group,^□□P<0.01 and^□P<0.05 vs ABMR+Tac group。

As can be seen from fig. 2, NL101 significantly reduced serum DSA levels in ABMR responses. Specifically, after 2 weeks of sensitization, IgG (P <0.001) and IgM (P <0.001) were significantly elevated after heterotypic kidney transplantation compared to homotypic kidney transplanted mice; daily tacrolimus administration reduces IgG and IgM produced by xenogenic kidney transplant rejection; however, after a single treatment with NL101 of heterotypic kidney transplantation, both IgG and IgM levels were significantly reduced, suggesting that DSA abundance was significantly reduced by NL 101.

3. Test result of endothelial C4d of transplanted tissue

C4d (green) immunoblot chromatograms in frozen sections of mouse kidney tissues from antigen sensitized group (Blank), homo-kidney transplantation group (Iso for short), heterotypic kidney transplantation resulting in acute ABMR group (ABMR), ABMR + tacrolimus treated group (ABMR + Tac) and ABMR + NL101 treated group (ABMR + NL101), and cell nuclei (blue) were stained with DAPI; the glomerular region is marked by a white circle (Bar is 100 μm, and the horizontal line in the figure indicates that the length is 100 μm, and is indicated by a scale).

After 2 weeks of sensitization, after 3 days of kidney transplantation of the homotypic kidney transplantation mice, the C4d (green) signal of the peripheral capillary endothelium in the tube of the kidney tissue is weaker, is related to the endothelial injury signal caused by ischemia reperfusion injury, belongs to non-DSA complement activation, and also accords with the result that B cells and plasma cells are increased in the flow cytometry result; signals of peripheral capillary vessel parts and glomerular capillary endothelium C4d in kidney tissues after 3 days of kidney transplantation of the heterotypic kidney transplantation mice are obviously enhanced; compared with the ABMR group, the daily administration of the tacrolimus can generally reduce the signal intensity of C4d, but the signal distribution is not changed; NL101 treatment resulted in a significant decrease in C4d signal, indicating that complement activation was significantly reduced by NL 101.

4. Measurement of inflammatory cell count in graft

FIG. 4A is a flow cytometric analysis of the transplanted kidneys of orthotopic kidney, homo-kidney transplant (Iso for short), allo-kidney transplant resulting in acute ABMR (ABMR), ABMR + tacrolimus treatment (ABMR + Tac) and ABMR + NL101 treatment (ABMR + NL101) mice of antigen sensitized (Blank) mice with CD45+ positive cells as a percentage of all cells; FIG. 4B is a statistical plot of the percentage of CD45+ positive cells in total cells of kidney (Blank group) or transplanted kidney (other group), wherein<0.0001,and **P<0.01 vs Blank group,^###P<0.001 and^#P<0.05 vs Iso group,^ΔΔΔP<0.001, and^ΔΔP<0.01 vs ABMR group。

Flow cytometry data showed that CD45+ cells accounted for approximately 7.98 ± 0.95% of the total number of cells in the homograft kidneys and were associated with ischemia-reperfusion injury; the percentage of CD45+ cells in total cells increased dramatically to 64.38 ± 4.81% in xenografted kidneys, demonstrating massive recruitment of inflammatory cells in the ABMR response; the percent of CD45+ cells in the transplanted kidney in the total cell number is down-regulated to 28.47 +/-6.88% (P <0.001 vs ABMR group) in the daily tacrolimus administration group, the percent of CD45+ cells in the transplanted kidney in the heterotypic kidney transplantation + NL101 treatment group is 18.22 +/-2.13%, the percent of CD45+ cells in the transplanted kidney in the total cell number can be obviously reduced (P <0.001), and the NL101 can reduce the recruitment of inflammatory cells in the transplant; taken together, NL101 was able to reduce the number of infiltrating graft inflammatory cells in the ABMR response.

5. Test results of local inflammatory factor level and circulating inflammatory factor level of graft

For antigen sensitized group (Blank), homo-kidney transplant group (Iso for short), and heterotypic kidneyThe serum levels of IL-6, IL-10, MCP-1, IFN-. gamma., TNF and IL-12p70 in mice from the acute ABMR group (ABMR), the ABMR + tacrolimus-treated group (ABMR + Tac) and the ABMR + NL 101-treated group (ABMR + NL101) resulting from transplantation were examined and counted, and the results are shown in FIG. 5 (A); the levels of IL-6, IL-10, MCP-1, IFN-. gamma., TNF, and IL-12P70 in supernatants of orthotopic kidney and other transplanted kidney tissues of antigen-sensitized (Blank) mice were measured and counted, and the results are shown in FIG. 5(B)<0.0001，***P<0.001,and*P<0.05 vs Iso group,^####P<0.0001，^###P< 0.001，^##P<0.01 and^#P<0.05 vs ABMR group,^ΔΔΔP<0.001,^ΔΔP<0.01 and^ΔP< 0.05 vs ABMR+Tac group。

In FIG. 5(A), the serum test results of mice showed that the level of IL-12p70 in the serum of the kidney transplant mice with the same type was higher than that of the blank control group, but there was no significant difference between the two values, so that the differences could be regarded as the injury changes associated with ischemia-reperfusion injury; IL-6, IL-10, MCP-1, IFN-gamma, TNF and IL-12P70 levels were all significantly increased in sera of heterotypic kidney transplant mice compared to homotypic kidney transplant kidneys, and tacrolimus and NL101 treatment significantly down-regulated each inflammatory factor, with NL101 further down-regulating IFN-gamma (P <0.05) compared to tacrolimus.

In FIG. 5(B), the results of the examination of mouse kidney tissue show that the levels of MCP-1 and TNF in the homograft kidney were increased on average, but did not show significant differences, compared to the blank group, and therefore these differences could be considered as the changes in injury associated with ischemia-reperfusion injury; the levels of IFN-gamma, IL-6 and MCP-1 in the heterotypic transplanted kidney are obviously increased compared with the levels in the homotypic transplanted kidney, and the levels of IL-10 and IL-12 are obviously reduced compared with the levels in the homotypic transplanted kidney; tacrolimus and NL101 treatment can significantly down-regulate IFN-gamma, IL-6, MCP-1 levels, while NL101 can up-regulate IL-10 levels. Taken together, NL101 was able to reduce graft local inflammatory factor levels and circulating inflammatory factor levels in ABMR responses.

6. Mortality results in Chronic ABMR groups

In the chronic ABMR model, because the protokidney of the mouse is removed, the metabolism of the body depends on the function of transplanted kidney, so if the kidney rejection occurs, the mouse can die due to uremia. The results in fig. 6 show that in Iso group, all mice survived successfully for more than 4 weeks, while in chrono ABMR group, mice developed rejection, with mortality reaching 80% in 1 week and total death in 2 weeks; rejection was better suppressed in mice treated with tacrolimus, mice began to die after 1 week and died 1 after 11 and 23 days, respectively, with 40% of mice surviving after 4 weeks; in the NL101 treated group, one mouse died after 11 days, and 60% survived after 4 weeks. This demonstrates that NL101 administration can significantly reduce the adverse effects of chrononic ABMR.

It should be understood by those skilled in the art that various features of the above-described embodiments can be combined in any combination, and for the sake of brevity, all possible combinations of features in the above-described embodiments are not described in detail, but rather, all combinations of features which are not inconsistent with each other should be construed as being within the scope of the present disclosure.

The above-mentioned embodiments only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application.