Application of B cell and IL-10 in preparation of product for treating osteoarthritis

Abstract

The invention discloses application of a B cell and IL-10 in preparation of a product for treating osteoarthritis. According to the application disclosed by the invention, a B cell deficient mouse, an IL-10 deficient mouse and a bone marrow remodeling technology are respectively utilized to reveal the specific action and the participation mechanism of the B cell in osteoarthritis development, and specifically, the B cell deficiency is proved to aggravate osteoarthritis lesion and cartilage destruction by utilizing a genetically deficient B cell mouse; meanwhile, the deficiency of the functional factor IL-10 in the B cell also aggravates the osteoarthritis lesion and cartilage destruction. The influence of injection of the B cells or the IL-10 into the articular cavity on osteoarthritis progress and cartilage destruction is further studied, and the study result shows that the injection of the B cells or the IL-10 into the articular cavity can effectively relieve OA progress and cartilage destruction and promote cartilage regeneration. The results show that the clinical value of the B cell or IL-10 in treatment of osteoarthritis is given, which is of great significance for developing a novel treatment strategy of osteoarthritis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of B cells and IL-10 in preparation of products for treating osteoarthritis. BACKGROUND

[0002] Osteoarthritis is a chronic degenerative joint disease characterized by progressive degeneration of articular cartilage, remodeling of subchondral bone, synovial inflammation and formation of marginal osteophytes, often accompanied by joint pain, stiffness and limited mobility, and can even lead to joint deformity and loss of function in severe cases. It is one of the main causes of disability in adults. The pathogenesis of osteoarthritis is complex and has not been fully elucidated. It is currently believed to be the result of multiple factors, including aging, abnormal mechanical stress distribution, abnormal remodeling of subchondral bone, metabolic imbalance of chondrocytes, synovial inflammation and immune microenvironment disorders (such as macrophages, T cells and B cells).

[0003] Currently, existing therapies for osteoarthritis aim to relieve symptoms and slow progression: non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics (such as acetaminophen) can provide short-term pain relief but cannot stop cartilage degeneration; intra-articular injection of sodium hyaluronate or glucocorticoids can temporarily improve joint lubrication and inflammation, but the duration of effect is short, and repeated injections may increase the risk of infection; end-stage patients require joint replacement surgery, which can significantly improve function but has problems such as surgical trauma, limited lifespan of prostheses and postoperative complications. Overall, existing therapies lack specific means to address the etiology and cartilage repair of osteoarthritis, and cannot fundamentally stop or reverse the disease progression.

[0004] B cells can participate in the development of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and other inflammatory diseases by antigen presentation, antibody secretion and release of TNF-alpha, IL-6, MMPs and other pro-inflammatory factors. In recent years, targeted B cell strategies represented by CAR-T cell therapy have shown significant results in new immunotherapy for SLE and RA, fully demonstrating their therapeutic potential. Studies have found that B cell infiltration increases in the peripheral blood and synovial tissue of patients with osteoarthritis, and there are abnormalities in proliferation and differentiation and changes in antibody secretion function, but it is not clear what role B cells play in osteoarthritis and what mechanisms regulate them, which to some extent restricts the development of new immunotherapy targeting B cells for osteoarthritis. Therefore, it is crucial to clarify the specific role and mechanism of B cells in the progression of osteoarthritis and further develop strategies that can effectively delay the progression of osteoarthritis and restore the structure and function of articular cartilage. SUMMARY

[0005] In view of the problems in the prior art, the application provides application of B cells and IL-10 in preparation of products for treating osteoarthritis.

[0006] To achieve the above object, the present application adopts the following technical solutions.

[0007] The first aspect of the present application provides the use of B cells in the preparation of a product for treating osteoarthritis.

[0008] The second aspect of the present application provides the use of IL-10 in the preparation of a product for treating osteoarthritis.

[0009] Preferably, the product is a drug.

[0010] The present application has the following beneficial effects:

[0011] The present application reveals the specific role of B cells in the progression of osteoarthritis and its participation mechanism by using B cell-deficient mice, IL-10-deficient mice, and bone marrow remodeling technology. Specifically, using mice with genetically deleted B cells, it is confirmed that B cell deletion aggravates osteoarthritis and cartilage destruction. At the same time, deleting functional factor IL-10 in B cells also aggravates osteoarthritis and cartilage destruction. The present application further clarifies the application value of intra-articular injection of B cells and its key functional factor IL-10 in the treatment of osteoarthritis. The research results show that injecting B cells or exogenous supplementing IL-10 into the joint cavity can significantly alleviate the progression of OA and cartilage destruction, and promote cartilage regeneration. The above results show the clinical value of giving B cells or supplementing IL-10 in the treatment of osteoarthritis, which has important significance for developing new treatment strategies for osteoarthritis. BRIEF DESCRIPTION OF DRAWINGS

[0012] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0013] Figure 1 Results of increased B cell infiltration in osteoarthritis (KOA) animal models: (a) experimental mode diagram: C57BL / 6J mice were used to establish a KOA model by ACLT, and the synovial tissue of the knee joint of the mice was taken after 8 weeks for flow cytometry analysis; (b) CD19 + B220 + B cell flow chart; (c) CD19 + B220 + B cell percentage and absolute cell number statistics chart;

[0014] Figure 2Results of B cell deficiency accelerating OA progression and cartilage destruction: (A) Experimental model diagram: C57BL / 6J mice (WT) and BCD mice were used to establish the KOA model by ACLT, and gait analysis, micro-CT and pathological staining (HE / Safranin O fast green staining) analysis were performed after 8 weeks; (B) Gait analysis results (walking time, walking speed and touch bottom coefficient) of WT mice and BCD mice after 8 weeks of KOA model establishment; (C) Micro-CT results and bone hyperplasia scores of WT mice and BCD mice after 8 weeks of KOA model establishment; (D) Pathological staining results of WT mice and BCD mice after 8 weeks of KOA model establishment; (E) Mankin score and OARSI score performed by blind observers showed that the KOA cartilage lesions of BCD mice were significantly aggravated;

[0015] Figure 3 Schematic diagram of bone marrow remodeling process of Breg functionally deficient mice;

[0016] Figure 4 Results of B cell specific IL-10 deficiency accelerating OA progression and cartilage destruction: (A) Gait analysis results (walking time, walking speed and touch bottom coefficient) of "WT" mice (BMC (BCD+WT)) and B cell specific IL-10 deficient mice (BMC (BCD+IL-10 - / - )) after 8 weeks of KOA model establishment; (B) Micro-CT results of "WT" mice (BMC (BCD+WT)) and B cell specific IL-10 deficient mice (BMC (BCD+IL-10 - / - )) after 8 weeks of KOA model establishment; (C) Bone hyperplasia score histogram; (D) Pathological staining results of "WT" mice (BMC (BCD+WT)) and B cell specific IL-10 deficient mice (BMC (BCD+IL-10 - / - )) after 8 weeks of KOA model establishment; (E) Mankin score and OARSI score performed by blind observers showed that the KOA cartilage lesions of (BMC (BCD+IL-10 - / - )) mice were significantly aggravated;

[0017] Figure 5 Comparison of flow cytometry before and after purification of naive B cells;

[0018] Figure 6Results of intra-articular B cell replenishment to alleviate OA progression and cartilage destruction: (A) Gait analysis results (walking time, walking speed, and touch bottom coefficient) of WT mice and BCD mice after KOA model establishment and B cell replenishment; (B) microCT results and bone hyperplasia score histogram of WT mice and BCD mice after KOA model establishment and B cell replenishment; (C) pathological staining results of WT mice and BCD mice after KOA model establishment and B cell replenishment; (D) OARSI score histogram of WT mice and BCD mice after KOA model establishment and B cell replenishment;

[0019] Figure 7 Results of intra-articular IL-10 replenishment to alleviate OA progression and cartilage destruction: (A) Gait analysis results (walking time, walking speed, and touch bottom coefficient) of WT mice and BCD mice after KOA model establishment and IL-10 replenishment; (B) microCT results of WT mice and BCD mice after KOA model establishment and IL-10 replenishment; (C) bone hyperplasia score histogram; (D) pathological staining diagram of WT mice after KOA model establishment and IL-10 replenishment; (E) Mankin score and OARSI score histogram of WT mice after KOA model establishment and IL-10 replenishment; (F) pathological staining diagram of BCD mice after KOA model establishment and IL-10 replenishment; (H) Mankin score and OARSI score histogram of BCD mice after KOA model establishment and IL-10 replenishment. DETAILED DESCRIPTION

[0020] In the following description, for purposes of explanation and not limitation, specific details are set forth such as particular architectures, technologies, techniques, etc. in order to provide a thorough understanding of the embodiments of the present application. However, it will be apparent to those skilled in the art that the present application can be practiced in other embodiments that depart from these specific details.

[0021] Embodiment 1

[0022] 1. Experimental method

[0023] (1) Establishing a knee osteoarthritis (KOA) animal model

[0024] First, the mice were anesthetized by intraperitoneal injection of Xylazine 50 (50 mg / kg), and then the OA mouse model was surgically induced by anterior cruciate ligament transection (ACLT): after shaving and disinfecting, the internal structure was exposed by gently slicing the outer skin and muscle tissue with a sterile surgical blade, the medial collateral ligament was cut after being found, and the entire joint cavity was opened; the anterior cruciate ligament was cut; the anterior drawer test was performed to confirm whether the anterior cruciate ligament was completely broken; after hemostasis, the incision was sutured layer by layer with surgical sutures; after the operation, 200,000 units of penicillin were intramuscularly injected to each mouse for three days.

[0025] (2) Introducing B cell-deficient mice (BCD mice) to establish KOA animal models

[0026] By introducing B cell-deficient mice (BCD mice), KOA animal models were established in comparison with wild-type (WT) mice. After 8 weeks, the mice in each group were taken out of the SPF animal room for gait analysis, microCT scanning and pathological staining.

[0027] First, gait analysis was performed: the VisuGait animal visual gait analysis system (Shanghai Xinxin Information Technology Co., Ltd., product model: XR-FP101) was used to record animal behavior. Before gait recording, the mice were pre-trained for 3 days by crossing a 100 cm walkway. The gait of each group was recorded at 8 weeks after OA induction before surgery. Two researchers who were unaware of the study group calculated and analyzed the relevant gait indicators.

[0028] Subsequently, the mouse knee joints were removed and fixed in 4% neutral formaldehyde for 48 h. The NanVoxel-1000 MicroCT (Tianjin Sanying Precision Instruments Co., Ltd.) was used to scan the formalin-fixed mouse legs with a source voltage of 100 kV and a current of 120 μA, and the resolution was 10 μm. The scan images of each group were evaluated at the same threshold to achieve three-dimensional structure rendering of each sample. Finally, the CT three-dimensional images of the knee joints of each group were scored using the osteophyte formation score.

[0029] For histopathological staining, the mouse knee joints were fixed in 4% formaldehyde for 48 hours, and then decalcified with EDTA for 3 weeks. After decalcification, the tissue was removed from the decalcification solution, routinely paraffin-embedded, and tissue sections were prepared. Coronal sections of mouse knee joints were taken with a thickness of 4 μm and stained with hematoxylin-eosin (HE) and Safranin-O-Green (Safranin-O-Green). The morphological features of articular cartilage, subchondral bone and synovial tissue were scanned using the tissue in situ cell scanning analysis system (Xiamen Jianfa High Technology Co., Ltd., model: Pannoramic MIDI II). Two blind observers used Mankin score and OARSI score to systematically score to assess the severity.

[0030] (3) Flow cytometry analysis of B cells

[0031] The knee joint synovial tissue of mice was collected, the surface residual blood was washed with normal saline first, and then cut into small pieces in the immune cell culture medium, and the digestion solution (final concentration of 1 mg / mL collagenase type IV and 0.1 mg / mL DNase) was added and 37°C water bath digestion for 20-30 minutes, then placed on the shaker (1200 rpm) for normal temperature digestion for 60 minutes. After digestion, terminate digestion on ice for 5 minutes, grind the precipitate and filter, then centrifuge at 1000g at 4°C for 8-10 minutes, discard the supernatant, add 42% Percoll solution, mix, then slowly add 70% Percoll solution along the tube wall, then centrifuge at 1260g at room temperature for 32 minutes. Take the middle white membrane layer to a 15 mL centrifuge tube, add PBS to 13 mL, then centrifuge at 800g at 4°C for 10 minutes, discard the supernatant, add 100 uL (anti-CD16 / 32 to avoid non-specific binding) blocking solution, resuspend, and block at room temperature for 10 minutes. For cell surface marker staining, mix the antibodies in 30 uL FACS (fluorescence activated cell sorting) buffer (PBS containing 0.5% BSA and 0.05% NaN3), add to the cells, and incubate at 4°C for 30 minutes. Cytoflex LX or LSR Fortessa flow cytometer (BD Biosciences) and FlowJo are used for detection and data analysis. The antibodies used are PE anti-mouse CD45.2 (BioLegend, 1098078, 1:400), APC / cy7 anti-mouse CD19 (Ebioscience, 47-0193-82, 1:300), EF450 anti-mouse B220 (Ebioscience, 48-0452-82, 1:300).

[0032] (4) Establish Breg function deficiency mice (B cell specific deletion of IL-10) by bone marrow remodeling

[0033] Reference Figure 3Rag1- / - mice, Rag1 gene encodes a protein essential for B cell and T cell maturation, responsible for V(D)J rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) genes. Therefore, Rag1- / - mice (lack of mature T cells and B cells) produced by gene encoding received X-ray irradiation (5-6 gray) for bone marrow depletion. The next day, bone marrow cells from WT, IL10- / - and BCD mice were collected from the leg bones of mice. The bone marrow cells of WT or IL10- / - and BCD mice were mixed at a ratio of 1:4 and injected into Rag1- / - mice through the tail vein for bone marrow reconstruction. The bone marrow of 80% of BCD mice provided almost all other immune cells except B cells, while the bone marrow cells of 20% of IL10- / - mice provided immune cells lacking IL-10 secretion function. Although it can also provide almost all immune cells, it only accounts for 20%, so here a B cell-specific IL-10-deficient bone marrow chimeric mouse (BMC (BCD+IL-10- / -)) is artificially constructed. In order to eliminate the influence of irradiation and bone marrow remodeling, the same method was used to mix 20% of WT mouse bone marrow and 80% of BCD mouse bone marrow and then input into Rag1- / - mice to construct a "WT mouse" (BMC (BCD+WT)). After 6-8 weeks, the success of bone marrow reconstruction was evaluated by measuring the proportion of adaptive immune cells in peripheral blood. The reconstructed mice were used for experimental analysis 8 weeks after reconstruction.

[0034] 2. Using B cell-deficient mice, IL-10-deficient mice, and bone marrow remodeling techniques, the specific role of B cells in the progression of osteoarthritis and its participation mechanism were studied

[0035] (2.1) Study the effect of B cell-specific IL-10 deficiency on the progression of osteoarthritis

[0036] Specific steps: B cell immune deficiency mice were used, and the osteoarthritis model was induced by surgery (anterior cruciate ligament transection surgery, abbreviated as ACLT model), and the differences in osteoarthritis behavior, imaging and pathological progression between B cell-deficient mice and wild-type (WT) mice were compared and analyzed. The results are shown in Figure 2 .

[0037] Figure 2 The results show that the gait of B cell-deficient mice is significantly abnormal, the intra-articular osteophyte formation is more obvious, the degree of articular cartilage destruction is significantly aggravated, and the cartilage matrix degradation is increased, confirming that B cells have an endogenous protective effect in osteoarthritis, and B cell deficiency can accelerate the progression of osteoarthritis in mice.

[0038] (2.2) Study the effect of B cell-deficient immune cytokine IL-10 on the progression of osteoarthritis

[0039] B cell specific IL-10 knockout mice (i.e. B cells can normally differentiate but cannot secrete IL-10) were constructed by bone marrow remodeling technology, and were used as controls with "wild type" (BMC (BCD+WT)) mice. KOA animal models were induced, and after 8 weeks, the mice in each group were taken out of the SPF animal room for gait analysis, micro CT scanning and pathological staining. The results are shown in Figure 4 .

[0040] Figure 4 The results showed that, compared with the control group of mice, B cell specific IL-10 knockout mice showed behavioral abnormalities, imaging lesions and cartilage destruction consistent with B cell completely deleted mice. This result confirmed that B cells mediate protection against OA by secreting IL-10, and IL-10 is a key effector molecule for B cells to function.

[0041] 3. Clinical value of B cell or IL-10 administration in the treatment of osteoarthritis

[0042] (3.1) Study of the effect of intra-articular injection of B cells on the progression of osteoarthritis and cartilage destruction

[0043] First, KOA models were established in wild type (WT) mice and B cell deleted mice using surgery induction, and after 2 weeks, 1x10 7 B cells were injected intra-articularly per mouse per week; then, the spleen single cell suspension of normal C57BL / 6J mice was collected, and CD19+ purified Naive B cells were obtained using magnetic bead sorting (see Figure 5 ). After sorting, 97.2% of the cells were CD19+ Naive B cells. The cells were washed twice with PBS, resuspended in 50 μL PBS at 1x10 7 cells per mouse; finally, after iodophor disinfection of the mouse knee joint, the B cell containing PBS was injected into the intra-articular cavity of the mouse knee joint through a sterile microsyringe. The injection was performed once a week, and the control group was treated with the same volume of PBS. After 4 weeks of administration, the progression of the disease in the mice was observed. The mice in each group were taken out of the SPF animal room for gait analysis, micro CT scanning and pathological staining. The results are shown in Figure 6 .

[0044] Figure 6 The results showed that, by injecting B cells into the intra-articular cavity, it was found that it could significantly alleviate the progression of disease in wild type mice and B cell deleted mice, improve gait, reduce osteophyte formation, reduce cartilage destruction, and promote cartilage matrix synthesis.

[0045] (3.2) Study of the effect of intra-articular injection of recombinant IL-10 supplementation (exogenous) on the progression of osteoarthritis and cartilage destruction

[0046] The osteoarthritis model is established on wild type mice and B cell deficient mice, after 2 weeks, the experimental group is injected with recombinant IL-10 protein, the injection volume is calculated according to the dosage concentration of 10 μg / kg, the recombinant IL-10 protein powder is dissolved in PBS, the injection volume of each mouse is 50 μL, and the injection is performed every three days. After the knee joint of the mouse is disinfected with iodophor, the PBS containing B cells is injected into the joint cavity of the knee joint of the mouse through a sterile microsyringe. The injection is performed once a week, the control group is treated with the same volume of PBS, and the disease progression of the mouse is observed after 4 weeks of administration. The mice in each group are taken out of the SPF animal room, and gait analysis, microCT scanning and pathological staining are performed, and the results are shown in Figure 7 .

[0047] Figure 7 The results show that by injecting the recombinant IL-10 protein into the joint cavity, it is confirmed that the (exogenous) supplement of IL-10 can effectively improve the OA pathological state, improve the gait, reduce the formation of osteophytes, reduce the destruction of cartilage, and promote the synthesis of cartilage matrix.

[0048] The present application is not limited to the above specific embodiments, and various modifications made by those skilled in the art without creative labor, all of which fall within the scope of the present application.

Claims

1. Use of B cells in the manufacture of a product for the treatment of osteoarthritis.

2. Use of IL-10 in the manufacture of a product for the treatment of osteoarthritis.

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