CN111662972A

CN111662972A - TRAF3 for preventing and treating arthritis by inhibiting IL-17 signal

Info

Publication number: CN111662972A
Application number: CN202010350036.8A
Authority: CN
Inventors: 张晓玲; 胡国立; 张宁
Original assignee: XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
Current assignee: XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2020-09-15

Abstract

The invention provides an application of an active ingredient, which is characterized in that: the active ingredients are selected from: (a) TNF receptor-related factor 3(TRAF 3); (b) a precursor capable of being processed in a host to TRAF3 in (a); (c) a polynucleotide capable of being transcribed by a host to form the precursor in (b) and processed to form TRAF3 in (a); (d) an expression vector comprising TRAF3 in (a), or the precursor in (b), or the polynucleotide in (c); the application of the active ingredients comprises the following steps: (a) the active ingredient is for inhibiting IL-17 signaling; (b) the active ingredient is used for preparing a medicament for preventing and treating arthritis.

Description

TRAF3 for preventing and treating arthritis by inhibiting IL-17 signal

Technical Field

The invention relates to an active factor, in particular to TRAF3 for preventing and treating arthritis by inhibiting IL-17 signals.

Background

Arthritis is the most common joint disease, most commonly Osteoarthritis (OA) and Rheumatoid Arthritis (RA), characterized by cartilage destruction, subchondral bone sclerosis and synovial inflammation. These abnormalities are mainly due to an imbalance between anabolism and catabolism of the articular cartilage, in particular an increase in catabolism. Proinflammatory cytokines, important mediators of arthritis, such as interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), and interleukin-6 (IL-6), are widely studied in articular cartilage. IL-17 is an important proinflammatory factor in the pathogenesis of a variety of autoimmune diseases such as Experimental Autoimmune Encephalomyelitis (EAE), collagen-induced arthritis (CIA), Inflammatory Bowel Disease (IBD). IL-17 is a major inflammatory driver cytokine that functions by inducing and maintaining the production of inflammatory cytokines, chemokines and matrix metalloproteinases. IL-17 may also act synergistically with IL-1 β or TNF- α to further induce pro-inflammatory genes. IL-17, a synovial fibroblast cell in Rheumatoid Arthritis (RA), has proven to be important in inducing the production of inflammatory factors interleukin and interleukin-8 (IL-8) through NF-. kappa.B and pi3 kinase/Akt signaling pathways. In OA, IL-17 is an upstream factor that regulates IL-1. beta. and TNF-. alpha. In articular cartilage and chondrocytes, IL-17 upregulates the mRNA expression of several cartilage catabolic factors such as interleukin 6(IL-6), Nitric Oxide (NO) and Matrix Metalloproteinases (MMPs) via NF-. kappa.B and MAPK signaling pathways. IL-17 is also more active in chondrocytes than in fibroblasts during OA pathophysiology. Interestingly, Th17 cells were significantly increased in anti-TNF α treated patients. Thus, simultaneous blockade of TNF- α and IL-17 in experimental arthritis was more effective than monotherapy. Therefore, blocking the IL-17 pathway is a promising approach to prevent cartilage degeneration in arthritis, either alone or in conjunction with current clinical arthritis therapies.

Tumor necrosis factor receptor-associated factors (TRAFs) are a family of intracellular adaptation proteins that mediate the downstream signaling of a variety of cell surface receptors, thereby controlling the early stages of cellular defense mechanisms against invading bacterial and viral pathogens. Most members of this family, including TRAF2, TRAF5 and TRAF6, have been extensively studied, however, TRAF3 has been rarely studied.

Disclosure of Invention

The invention aims to overcome the defects, the function and the mechanism of TRAF3 are researched, and TRAF3 is found to be a potential therapeutic target for improving the cartilage degeneration of arthritis.

The invention provides an application of an active ingredient, which is characterized in that:

the active ingredients are selected from:

(a) TNF receptor-related factor 3(TRAF 3);

(b) a precursor capable of being processed in a host to TRAF3 as described above in (a);

(c) a polynucleotide capable of being transcribed by a host to form the precursor described above in (b) and processed to form TRAF3 described above in (a);

(d) an expression vector comprising the TRAF3 described above in (a), or the precursor described above in (b), or the polynucleotide described above in (c);

the application of the active ingredients comprises the following steps:

(a) the active ingredient is for inhibiting IL-17 signaling;

(b) the active ingredient is used for preparing a medicament for preventing and treating arthritis.

Further, the invention provides an application of the active ingredient, which is characterized in that: the above active ingredients are used for preparing medicines for treating, preventing, improving or relieving synovitis, cartilage injury or cartilage degeneration caused by arthritis.

The above active ingredient is used in an effective amount, and generally, the amount can be adjusted according to the conventional formulation of the pharmaceutical preparation. Such as: the dosage of the preparation is 10^-8-100％。

Further, the invention provides an application of the active ingredient, which is characterized in that: the medicine is in a dosage form of gastrointestinal administration or a dosage form of parenteral administration.

The above gastrointestinal administration dosage form is selected from powder, tablet, granule, capsule, solution, emulsion, and suspension;

the above-mentioned dosage form for parenteral administration is selected from injection dosage form, respiratory tract dosage form, nasal drop, skin dosage form, mucous membrane dosage form or cavity administration dosage form.

Further, the invention provides an application of the active ingredient, which is characterized in that: the active ingredients are also useful for interfering with the binding of IL-17R to Act 1.

Further, the invention provides an application of the active ingredient, which is characterized in that: the active ingredients are also useful for inhibiting IL-17 mediated NF- κ B and MAPK pathways, and reducing IL-6 and matrix metalloproteinase production.

Further, the invention provides an application of the active ingredient, which is characterized in that: the above active ingredients are also useful for inhibiting IL-17 mediated phosphorylation of p65, p38, JNK and ERK.

Further, the invention provides an application of the active ingredient, which is characterized in that: the above active ingredients are also useful for inhibiting IL-17 induced MMP-13 production.

In addition, the arthritis animal model is characterized by being selected from one or more of the following animal models:

(A) an IL-17-induced rodent comprising a modified, IL-17-induced rodent for use in constructing a model of arthritis with high expression of the inflammatory factor IL-17 in a joint;

(B) a DMM-induced rodent comprising a modified, animal model of osteoarthritis;

(C) a rodent comprising a modified rodent for use in the construction of an age-related animal model of idiopathic osteoarthritis.

Further, the invention provides an ulcerative colitis animal model, which is characterized in that:

the rodents mentioned in (B) or (C) are also induced by IL-17.

Further, the invention provides an ulcerative colitis animal model, which is characterized in that: the rodents described above trap a TRAF3 knockout rodent.

The invention has the following functions and effects:

the invention researches the action and mechanism of TRAF3, and finds that the up-regulation of TRAF3 expression in osteoclast precursor can reduce bone destruction and inflammatory bone loss of common bone diseases. Meanwhile, TRAF3 is a key factor controlling the expression of type I interferon-inducible and anti-inflammatory cytokine interleukin 10 (IL-10). These findings suggest that TRAF3 plays a complex role in restoring immune homeostasis.

In addition, the research also finds that TRAF3 can play a negative regulation role in IL-17 mediated NF-kB and MAPK signal paths, and the IL-17 signal path is greatly enhanced by knocking TRAF3 out of human primary synovial cells. Meanwhile, in the OA process, IL-17 affects chondrocytes more than synoviocytes.

Therefore, the invention considers that over-expressing TRAF3 in chondrocytes obviously inhibits IL-17 mediated NF-kB and MAPK pathways, thereby reducing the production of IL-6 and matrix metalloproteinase. In contrast, TRAF3 knock-out resulted in IL-17-induced elevation of matrix metalloproteinase-13 (MMP-13). In vivo, loss of IL-17 significantly reduced cartilage destruction caused by surgery-induced OA and age-related spontaneous OA. TRAF3 transgenic mice had less cartilage destruction in different animal models of arthritis compared to the control group. In addition, using adenovirus to silence TRAF3 significantly exacerbated cartilage degradation in experimental OA, and deletion of IL-17 partially rescued cartilage degradation in experimental OA. In summary, TRAF3 should be a potential therapeutic target for improving arthritic cartilage degradation.

Drawings

IL-17 activates factors that upregulate catabolism in chondrocytes by NF-. kappa.B and MAPK signaling.

Wherein, Panel A is the level of catabolic gene mRNA in IL-17 stimulated primary chondrocytes (24h,10 ng/mL);

FIG. B shows the level of catabolic gene mRNA in IL-17 stimulated ATDC5 cell line (24h,10 ng/mL);

FIG. C shows the level of catabolic gene mRNA in IL-17 stimulated SW1353 cell line (24h,10 ng/mL);

FIG. D is an immunoblot of major molecules involved in NF-. kappa.B and MAPK signaling pathways in IL-17 mediated primary chondrocytes;

FIG. E is an immunoblot of major molecules involved in NF-. kappa.B and MAPK signaling pathways in the IL-17 mediated ATDC5 cell line;

FIG. F immunoblot of major molecules involved in NF-. kappa.B and MAPK signaling pathway in IL-17 mediated SW1353 cell line;

panel G is a quantification of the immunoblot results shown in panel D. Data represent mean ± s.e.m. of at least 3 independent experiments;

panel H is a quantification of the immunoblot results shown in panel E. Data represent mean ± s.e.m. of at least 3 independent experiments;

FIG. I is a quantification of the immunoblot results shown in FIG. F. Data represent mean ± s.e.m. of at least 3 independent experiments;

***p<0.05,p<0.01,***p<0.001。

FIG. 2A. TRAF3 expression levels of Ad-Traf3 infection in primary chondrocytes;

FIG. 2B immunoblot of Ad-Traf3 infected TRAF3 in primary chondrocytes;

FIG. 3 TRAF3 negatively regulates il-17 mediated signal transduction and induction of matrix degrading enzymes in chondrocytes.

Wherein, Panel A is an immunoblot of primary chondroblasts transfected with plasmid Empty Vector (EV) or TRAF3 labeled M2(Flag) with anti-p65, anti-p-p65anti-p-JNK, anti-p38, anti-p-p38, anti-ERK, anti-p-ERK, anti-TRAF3 or anti- β -actin;

FIG. B is an immunoblot of intact cell lysates of ATDC5 cells untreated or treated with IL-17(10ng/mL) for 15 or 30min with anti-p65, anti-p-p65anti-p-JNK, anti-p38, anti-p-p38, anti-ERK, anti-p-ERK, anti-TRAF3 or anti- β -actin;

panel C shows IL-17(10ng/ml) induced expression of IL-6, MMP-13, ADAMTS-4, ADAMTS-5mRNA in Ad-C or Ad-Traf3 infected primary chondrocytes;

FIG. D is IL-17(10ng/ml) induced expression of IL-6, MMP-13, ADAMTS-4, ADAMTS-5 mRNAs in Ad-C or Ad-Traf3 infected ATDC5 cells;

FIG. E is an immunoblot analysis of catabolic factors (MMP-13 and COX-2) from primary chondrocytes infected with Ad-C or Ad-Traf3 using Western blot analysis;

FIG. F is an immunoblot analysis of catabolic factors (MMP-13 and COX-2) in primary chondrocytes infected with Ad-C or Ad-sh-Traf 3;

panel G is a quantification of MMP-13 immunoblot results shown in panel E, data representing mean ± s.e.m. of at least 3 independent experiments;

panel H is a quantification of COX-2 immunoblot results shown in panel E, data representing mean ± s.e.m. of at least 3 independent experiments;

panel I is a quantification of MMP-13 immunoblot results shown in panel F, data representing the mean ± s.e.m. of at least 3 independent experiments;

panel J is a quantification of COX-2 immunoblot results shown in panel F, data representing mean ± s.e.m. of at least 3 independent experiments;

***p<0.05,p<0.01。

fig. 4a. effect of TRAF3 expression on mRNA and protein levels in primary chondrocytes;

fig. 4b immunoblot of TRAF3 expressed mRNA and protein levels in primary chondrocytes;

FIG. 5A.3 Effect of Act 1-specific siRNAs on Act1 in chondrocytes;

FIG. 5B.3 immunoblotting of Act1 in chondrocytes with Act 1-specific siRNAs;

FIG. 6 Effect of knockout Act1 on IL-17 induced MMP-13 protein levels;

FIG. 7. the absence of IL-17 attenuates surgical OA-induced and senescence-induced degradation of cartilage matrix in spontaneous OA;

wherein panel a is the cartilage destruction and OARSI rating (n 6) at 3 months of age for medial meniscus (DMM) operated WT and IL-17 a-/-mice;

panel B shows safranin-O staining and OARSI grading (n-8) for 14 month WT and IL-17 a-/-mice, scale bar: 50 μm, values representing mean ± s.e.m.. p < 0.01.

FIG. 8. less IL-17 induced MMP13 production and cartilage EMC loss in TRAF3 transgenic mice (T3 TG);

wherein panel a is an immunohistochemical representative image of MMP-13 following intraarticular injection of IL-17 or PBS in T3TG (n-6) and littermate control (n-6) mice;

panel B shows immunohistochemical quantitation of MMP-13 following intraarticular injection of IL-17 or PBS in T3TG (n-6) and littermate control (n-6) mice on a scale of 50 μm and 20 μm in the frame. MMP-13 positive chondrocytes are shown in frame;

panel C shows T3TG cartilaginous safranin-O staining (n-6) and littermate control (n-6) after intra-articular injection of IL-17 or PBS, scale bar 50 μm, in-frame area scale bar 20 μm. The boxed area shows articular cartilage enlargement, values mean ± s.e.m.. p <0.05, p <0.01,. p < 0.001.

FIG. 9 reduction of TRAF3 expression during the onset of experimental OA;

wherein, panel a is the immunohistochemistry results of TRAF3 expression in DMM-induced experimental OA or Sham group rats 1 month (n-8), 2 months (n-6), 3 months (n-9) cartilage after surgery, scale bar: 20 μm;

panel B shows the change in the expression level of TRAF3 mRNA in rat cartilage tissue as described above;

panel C shows TRAF3 positive chondrocytes (n: 3) in articular cartilage tissue of OA patients, scale bar: 50 μm, scale bar of the area in frame: 20 μm, and the area in frame shows TRAF3 positive chondrocytes, with values expressed in terms of ± s.e.m.. p <0.05, p <0.01,. p < 0.001.

FIG. 10 TRAF3 might control the development of OA by inhibiting IL-17 signaling;

wherein panel a is WT and T3TG mouse sham surgery or unstable cartilage destruction and OARSI grading (n 12) for medial meniscus (DMM) procedure with scale bar 100 μm, in-frame area scale bar 50 μm, in-frame area showing articular cartilage enlargement;

panel B shows safranin-O staining and OARSI grading (n 10) of articular cartilage obtained by intra-articular injection of Ad-C or Ad-sh-Traf3 in sham or DMM-operated WT or IL-17 a-/-mice (scale bar: 50 μm, values representing mean ± s.e.m. p <0.05, p < 0.01).

Detailed Description

First, materials and experimental methods:

A. animal(s) production

TRAF3 transgenic mice and IL-17 a-/-mice were given by professor Sucare of Sum Youthow, Shanghai Life sciences institute of Chinese academy of sciences. The generation of TRAF3 transgenic mice was reported in previous studies. T3TG and IL-17 a-/-mice and 8-12 week old littermates wild type mouse (WT) control groups were used in the experiments. C57BL/6 mice and SD rats were purchased from Shanghai laboratory animal center, Chinese academy of sciences. All animals were kept under sterile conditions. All animal experiments were performed according to the guidelines for laboratory animal feeding and use and were approved by the ethical committee of biomedical research of the Shanghai Life sciences institute of Chinese academy of sciences.

Model of IL-17 intra-articular injection

Mice were anesthetized with 250mg/kg tribromoethanol (Sigma, St. Louis, Mo., USA). A total amount of 10ng/ml recombinant IL-17 (McOmn MN, developed in the United states) was included in 5 μ l in PBS and injected as described previously through the infrapatellar ligament into the left knee space with a 30G needle (65460-05, Hamilton, Inc., Switzerland) with the patellar ligament visible through a small incision in the skin and the right knee injected with 5 μ l PBS as a control. IL-17(10ng/ml in PBS) and PBS were injected into T3TG mice and littermate WT control groups, respectively, 3 consecutive injections every 24 hours. Knee joint specimens were collected 3 days after the last injection of T3TG and WT mice were treated with CO2 (10 per group) and fixed with 4% paraformaldehyde.

A-2.DMM model and OARSI score

Instability of the mouse medial meniscus (DMM) was caused by transection of the anterior attachment portion of the medial meniscus to the tibial plateau. The knee was then prepared for sterile surgery, a longitudinal incision was made medial to the patellar ligament, the joint capsule opened, and the meniscal ligament securing the medial meniscus to the tibial plateau was discovered. A portion of the animals in the sham group were not subjected to further procedures. The experimental group severed the medial meniscal ligament to form DMM. The prosthetic surgery group and the DMM group suture the joint capsule and the subcutaneous layer, respectively. T3TG and WT mice were euthanized by CO2 treatment 6 weeks after DMM. For adenovirus injection, control group virus (Ad-C) or Ad-sh-TRAF3 virus was injected intra-articularly 1 time for 5 weeks within one week after DMM surgery. The knee joints were collected, left to stand overnight at 4 ℃ in 10% formalin buffer, and decalcified with 14% EDTA for 2 weeks. The joint was embedded in paraffin and cut 5 μm thick. Histological sections were prepared, stained with reddish-O and fast green, and then graded for OA severity by 4 independent scorers using the international association for osteoarthritis research (OARSI) scoring system. Three sections (medial, central, lateral) were taken from each joint for staining and evaluation. The three segments are classified separately, and the average value is taken for each segment.

A-3. rat OA animal model human cartilage specimen

As described above, DMM surgery induced OA in rats. Briefly, the animals are anesthetized and then surgery is performed to transect the MCL. The medial meniscus was dissected full thickness to induce destabilization of the right knee. In the sham group, no ligament transection or meniscus tear occurred, as in the control group. The patients are sacrificed after 1, 2 and 3 months of operation (7-10 patients in each group), knee joint specimens are collected, and fixed by 4% paraformaldehyde. OA cartilage of a patient who underwent total joint replacement due to primary knee osteoarthritis was sampled (N ═ 3). The same patient was treated as normal cartilage in the intact area. Ethical approval was obtained from the medical ethics committee of the ninth national hospital, shanghai university of transportation, and informed consent was obtained from all participants.

A-4. histology and immunohistochemistry

The knee joints of the experimental animals were fixed overnight with 12.5% EDTA decalcified 4% paraformaldehyde PBS (pH 7.0) and paraffin embedded. Tissue sections (5 μm) were deparaffinized in xylene, hydrated continuously with ethanol, and rinsed with PBS. Sections were stained with reddish-O/fast green to identify proteoglycan loss. The reddish-O staining was quantified by density using Image J software (Image J1.52 t, https:// Image J. nih. gov/ij /). In immunohistochemistry, tissue sections were heated in 10mm sodium citrate buffer (pH 6.0) at 95 ℃ for 10 minutes in a microwave oven. The sections were cooled at room temperature for 30 minutes before antigen exposure. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide and washed several times with PBS. After blocking 5% BSA and non-specific proteins in PBS for 30min at room temperature, the cells were incubated overnight at 4C with primary antibodies to TRAF3(Abcam, ab217033, Cambridge, UK) or MMP-13(Abcam, ab 219620). Washed with PBS and then incubated with secondary antibody (Cell signaling technology, Danvers, MA, USA) as per the manufacturer's instructions. Sections were then stained with hematoxylin (Sigma). After washing, staining was performed with 3, 3' -diaminobenzidine hydrochloride (EnVision detection kit, peroxidase/DAB, rabbit/mouse, Dako cytoma, Santa Clara, CA, USA). Normal IgG staining and no primary antibody staining also served as negative controls. The Image J software was used to calculate the percentage of positive cells and quantify the immunohistochemistry results.

B. Cell culture

B-1. isolation and culture of rat chondrocytes

Rat knee cartilage was isolated from rat chondrocytes, filtered 5 hours after collagenase type II digestion using a 70 μm filter (BD Corning, Franklin lake, New Jersey, USA), and then cultured in DMEM/F12 (texture of Dulbecco's modified Eagle's medium and Ham's F12 medium, Gibco BRL, Germany) with 10% FBS (fetal bovine serum, Gibco BRL, Germany), 100 units/ml penicillin G,100 μ G/ml streptomycin, and 2.5 μ G/ml amphotericin B, cultured in a 5% carbon dioxide environment at 37 ℃.

ATDC5 cell line B-2

Mouse cartilage-derived ATDC-5 cell lines were taken from the Riken cell bank of Wako, Japan. Undifferentiated ATDC5 cells were cultured in DMEM/F12 medium containing 10% FBS,100 units/ml penicillin G, 100. mu.g/ml streptomycin, 2.5/ml amphotericin B.

B-3.SW1353 cell line

Human chondrosarcoma cell line SW1353 cells were obtained from standard culture collection of chinese academy of sciences. Cells were also cultured in DMEM/F12.

B-4. transient transfection of Gene expression

The labeled TRAF3 was cloned into the retrovirus-mediated pMSCV-IRES-GFP vector (supplied by the Cinchonary laboratory of the health sciences institute of the Shanghai Life sciences, Chinese academy of sciences) and the pcDNA 3.0 plasmid. ATDC5 cells were infected with viral supernatant of 293FT cells transfected with TRAF3 plasmid and helper vector. SW1353 cells were transfected with pcDNA-TRAF3 and empty pcDNA vector using lipofectamine2000(Invitrogen, Carlsbad, Calif., USA).

RNA interference B-5

RNA interference was performed using siGENOME SMART pool siRNA (Dharmacon, Lafayette, CO, USA) targeted to rat Act1(gene ID 369048).

RNA purification and real-time fluorescent quantitative PCR (qRT-PCR)

Total RNA was prepared using TRIzol reagent (Invitrogen) as per the instructions. 1ug of total RNA was incubated with reverse transcriptase at 42 ℃ for 1 hour to synthesize the first strand of cDNA. After the reverse transcription reaction, qRT-PCR was performed using the LightCycler480 system (SYBR1Premix Ex TaqTM) (TakaraBio, Japan) manufactured by Roche of Switzerland according to the manufacturer's instructions. The real-time PCR was performed under the conditions of 95 ℃ 10s denaturation, followed by 95 ℃ 10s 40 cycles, and finally 60 ℃ 30 s. The lysis phase is added to the end of the amplification process until the lysis curve shows no non-specific amplification. All amplifications were referenced against beta-actin. Data analysis was performed using the comparative Ct (2- Δ Ct) method and expressed as a broken line variation.

B-7. immunoblotting

Cells were lysed in a lysate containing 50mm Tris-HCl, pH 7.4, 150mm NaCl, 1% Nonidet P-40, 0.1% SDS protease inhibitor (10mg/ml albumin, 10mg/ml pepstatin A and 10mg/ml aprotinin) on ice for 30 min. In immunoblot analysis, 50 μ g protein samples were run on 10% SDS-PAGE and transmembrane spun on nitrocellulose membranes (Whatman, Perscatavir, New Jersey, USA). The main antibodies used anti-TRAF3(1:1000) (Abcam) MMP-13 (1: sc-30073, Santa Cruis Biotech., Santa Cruis, CA, USA), cox-2(1:500, ab15191 Abcam) GAPDH (1:5000, G9545, Sigma), and anti-Phospho-p65(1:1000) anti-p65(1:1000) anti-Phospho-p38 MAPK (1:1000) anti-p38MAPK (1:1000) anti-Phospho-p44/42MAPK (1:2000), anti-p44/42MAPK (1:1000) anti-Phospho-SAPK/1: 1000) and anti-SAPK/1: 1000, beta-actin as controls. hrp conjugated secondary antibodies (cell signaling technique) were diluted 1: 1000. Antigen-antibody complexes were visualized using an enhanced chemiluminescence detection system (MILlipopore, BILlerica, MA, USA) according to the manufacturer's recommendations. Immunoreactive bands in 3 replicate samples were quantitatively analyzed by normalizing immunoreactive band intensities on the scanned membrane to beta-actin using Alpha image software.

B-8 statistical analysis

Statistical analysis was performed using GraphPad Prism software (v6, Durham, NC, https:// www.graphpad.com /). All data are expressed as mean ± Standard Deviation (SD). Statistical differences between the two groups were determined by the two-tailed student t-test. A P value less than 0.05 is statistically significant. All experiments were performed on at least 3 independent samples.

II, experimental results:

IL-17 Induction of inflammatory cytokine production by chondrocytes, MMPs and protease

To investigate the role of IL-17 in chondrocyte catabolism, chondrocytes were treated with recombinant IL-17(10ng/ml, 24 h). IL-17 treatment resulted in a significant upregulation of IL-6, MMP-13, a thrombospondin motif-4 (ADAMTS-4), and ADAMTS-5 at the mRNA level, which was critical in OA cartilage destruction in rat primary chondrocytes (shown in FIG. 1A), ATDC5 cells (shown in FIG. 1B), and SW1353 cells (shown in FIG. 1C).

In addition, phosphorylation of p65, p38, ERK and JNK was triggered immediately after treatment with IL-17 in rat primary chondrocytes (as shown in fig. 1D and 1G) and ATDC5 cells (as shown in fig. 1E and 1H).

However, IL-17 only activated phosphorylation of p65, ERK and JNK, and did not activate p38 in SW1353 cells (as shown in FIGS. 1F and 1I).

These data indicate that IL-17 upregulates chondrolytic metabolism factors by activating NF-. kappa.B and MAPK signaling pathways.

TRAF3 stringent control of IL-17 induced catabolic gene expression

To determine the role of TRAF3 in IL-17 mediated signal transduction, this example generated an adenovirus comprising the entire CDS sequence of TRAF3 (Ad-TRAF 3).

Indeed, Ad-TRAF3 infection in primary chondrocytes (as shown in fig. 2A and 2B) effectively increased TRAF3 expression levels.

It was also found in this example that overexpression of TRAF3 significantly inhibited IL-17-mediated phosphorylation of p65, p38, JNK and ERK, indicating that TRAF3 has a general inhibitory effect on IL-17-mediated signaling pathway (as shown in fig. 3A).

In addition, this example also transfected TRAF3 or an empty vector plasmid into SW1353 cells and tested for IL-17 mediated immediate signaling. It is agreed that overexpression of TRAF3 inhibited IL-17-induced phosphorylation of p65, p38 and JNK, but not ERK phosphorylation (fig. 3B), indicating that TRAF3 has a general inhibitory effect on IL-17-mediated pathways in chondrocytes.

Then, this example also investigated the role of TRAF3 in IL-17 induced cartilage catabolic factor expression in chondrocytes. Notably, overexpression of TRAF3 in primary chondrocytes and ATDC5 cells at the mRNA level (as shown in FIGS. 3C and 3D) inhibited IL-17-mediated upregulation of IL-6, MMP-13, Adamts-4, but not Adamts-5.

Overexpression of TRAF3 also significantly reduced protein expression of MMP-13 and cox-2 under IL-17 stimulation (FIGS. 3E,3G and 3H), to further confirm the role of TRAF3 in IL-17 mediated downstream gene induction, TRAF 3-specific siRNA (specific siRNA against TRAF3, 5' GAAGGTTTCCTTTGTTGCAGAATGAA-. The data indicate that TRAF3 expression is effective in reducing mRNA and protein levels in primary chondrocytes (as shown in fig. 4A and 4B). As expected, the loss of TRAF3 function enhanced IL-17-induced up-regulation of MMP-13 and COX-2 at the protein level (as shown in FIGS. 3F, 3I, and 3J).

Given that TRAF3 interferes with IL-17R binding to Act1, blocking the recruitment of TRAF6, ultimately leading to IL-17 mediated signaling pathways and inhibition of downstream gene transcription, this example generated 3 siRNAs specific for Act1 to knock down Act1 in chondrocytes. siRNA #3 was used in the following study because of its highest knockdown efficiency (as shown in fig. 5A and 5B).

Importantly, knock-out of Act1 significantly reduced IL-17-induced up-regulation of MMP-13 protein levels (as shown in figure 6). Taken together, these data strongly demonstrate that TRAF3 has a critical control effect on IL-17-induced expression of catabolic genes downstream of chondrocytes.

Experiments on IL-17 deficiency to protect cartilage degradation and spontaneous osteoarthritis

This example further investigates whether blocking IL-17 signaling can protect cartilage degradation in vivo. Interestingly, the cartilage destruction induced by DMM surgery was significantly reduced in IL-17 deficient mice compared to the control group (as shown in figure 7A). In addition, the reduction of IL-17 also significantly reduced cartilage degradation caused by age-related spontaneous OA (as shown in FIG. 7B). Taken together, these results indicate that IL-17 plays a key role in cartilage degradation during OA.

IL-17-induced less stroma loss in TRAF3 transgenic mice

To investigate the role of TRAF3 in the in vivo-induced cartilage degradation of IL-17, this example established an IL-17 intra-articular injection model using T3TG mice and a control group of littermate WT mice. MMP-13 is a key degradation factor for the loss and degradation of the chondrocyte extracellular matrix (ECM). It was found that after IL-17 injection, MMP-13 production was significantly increased in the superficial cartilage region of WT mice, while MMP-13 production was significantly inhibited in the cartilage of T3TG mice (as shown in FIGS. 8A and 8B). The loss of chondral ECM in T3TG mice resulting from IL-17 injection (which is reflected by reddening-O staining) was also reduced compared to the control group. Taken together, these results demonstrate that TRAF3 is able to attenuate IL-17-induced MMP-13 production, thereby inhibiting cartilage degradation in vivo.

Reduced expression of TRAF3 in OA cartilage

To investigate whether the expression level of TRAF3 in articular cartilage was altered during OA. This example also established an experimental OA rat model to study TRAF3 expression in OA and normal cartilage. Immunohistochemistry results showed that the OA cartilage TRAF3 expression level was slightly decreased at 1 month after surgery and significantly decreased at 2 and 3 months after surgery in the experimental group compared to the sham group (as shown in fig. 9A).

Meanwhile, the mRNA level of TRAF3 in cartilage of different stages of rat experimental OA model was analyzed in this example, and TRAF3 was found to be significantly reduced during OA (as shown in FIG. 9B).

Also, expression levels of TRAF3 were found to be lower in OA cartilage than in normal cartilage in human OA patients (as shown in fig. 9C).

Taken together, these observations suggest that TRAF3 is down-regulated in cartilage during the onset of OA.

TRAF3 protective in OA-induced cartilage degradation

Given the in vitro role of TRAF3 in inhibiting IL-17 mediated signal transduction and inducing matrix degrading enzymes, this example further investigated whether TRAF3 affects the pathogenesis of experimental OA in vivo. To this end, this example established an experimental OA model by DMM surgery in T3TG mice and a control group of littermate WT mice.

After 1 month of surgery, TRAF3 deficiency was found to be effective in relieving cartilage destruction induced by DMM surgery, both histology and OARSI scores were shown (as shown in fig. 10A). To further confirm whether TRAF 3-mediated inhibition of IL-17 signaling results in its protective effect on cartilage degradation, in this example, an adenovirus containing a TRAF3 short hairpin RNA plasmid (Ad-sh-TRAF3) was implanted into the knee joint of DMM-induced OA mice. The results show that deletion of TRAF3 aggravated cartilage destruction by surgically-induced OA, whereas Ad-sh-TRAF3 had no effect on cartilage degradation by IL-17 a-/-mouse DMM surgery (as shown in FIG. 10B).

Thus, it was shown that inhibition of TRAF 3-mediated IL-17 signaling is a potentially effective therapeutic target for OA cartilage matrix degradation.

Thirdly, discussion of results:

IL-17 has been found to activate a variety of signaling pathways, strongly inducing proinflammatory cytokines and chemical kinases. In addition, IL-17 may also act synergistically with IL-1 β or TNF- α to further induce proinflammatory cytokines, NO and MMPs, and to reduce proteoglycan levels. In fact, IL-17, an upstream factor, significantly increased the expression of IL-6, MMP-13, ADAMTS-4, and ADAMTS-5 in primary chondrocytes, the ATDC5 cell line, and the SW1353 cell line.

However, the up-regulation of these catabolic factors induced by IL-17 is relatively mild compared to IL-1 β or TNF- α, suggesting that IL-17 may not directly regulate these catabolic factors, and instead IL-17 may turn on the expression of these catabolic factors by up-regulating the expression of IL-1 β or TNF- α through activation of the NF-. kappa.B and MAPK pathways. Similarly, local administration of IL-17 in vivo induces MMP-13 production by articular cartilage, with concomitant loss of cartilage matrix. However, only mild stromal loss was observed in the IL-17 injection model, probably due to the shorter IL-17 exposure time (3 days). Most importantly, IL-17 mice with systemic loss of function exhibited reduced cartilage degradation in both surgically-induced OA and age-related spontaneous OA, suggesting that IL-17 may be an important mediator of cartilage degradation during OA. Therefore, blocking IL-17 mediated chondrocyte downstream signaling is an effective strategy to protect cartilage from degradation.

TRAF3 is considered to be an important negative regulator in the non-classical NF-. kappa.B pathway in a variety of cell types. Previous studies have shown that IL-17 stimulation induces the recruitment of TRAF3 to the IL-17 receptor (IL-17R), thereby interfering with the formation of the IL-17R-act1-TRAF6 activation complex. Previous studies have shown that TRAF3 can inhibit IL-17 mediated signal transduction in primary synovial cells. In addition, TRAF3 overexpression significantly inhibited IL-17 mediated phosphorylation of p65, JNK, ERK and p38, and thus produced IL-6, MMP-13, Adamts-4 and COX-2, but did not inhibit Adamts-5 in primary chondrocytes. However, overexpression of TRAF3 inhibited only p65 and JNK phosphorylation in ATDC5 cell line, suggesting heterogeneity of TRAF3 function in different cell types. To demonstrate the protective effect of TRAF3 on IL-17 mediated cartilage degradation in vivo, this example established an IL-17 intra-articular injection model. Importantly, the increased function of TRAF3 effectively reduced IL-17-induced MMP-13 production and matrix loss in mice. Interestingly, TRAF3 overexpression did not affect the expression levels of Adamts-5 in primary chondrocytes and the ATDC5 cell line. Adamts-5 is considered to be the major mouse protease, and thus, the functional gain of TRAF3 may not attenuate proteoglycan degradation in mice.

Both cartilage matrix loss and cartilage degradation were reduced in T3TG mice compared to wild type mice. Mild cartilage ECM loss (grade 1-2) was observed post-operatively in T3TG mice according to the OARSI score. No significant degradation of cartilage was seen in T3TG mice, and degradation of WT cartilage was evident (grade 2-5). Furthermore, T3TG mice (stage 1-2) had less effect on surface area than WT mice (stage 3-4). On the other hand, loss of TRAF3 function enhances cartilage destruction resulting from DMM surgery. Interestingly, the expression level of TRAF3 decreased significantly in the late phase of OA pathogenesis (starting from 2 months after DMM), while there was no significant decrease in the early phase of rat OA cartilage. At 3 months after DMM surgery, little TRAF3 was detected in the articular cartilage (fig. 9A). mRNA expression further confirmed significant changes in TRAF3 during OA, suggesting that TRAF3 is down-regulated in transcript levels during OA pathogenesis, leading to an enhanced IL-17 signaling pathway in cartilage. These results indicate that TRAF3 is a key negative regulator of cartilage degradation during OA. More importantly, deletion of the IL-17 gene blocked TRAF3 knock-out mediated cartilage destruction. These data indicate that the protective effect of TRAF3 on cartilage is achieved by inhibiting IL-17 mediated signal transduction.

In conclusion, this example demonstrates that TRAF3 plays an important role in protecting the degradation of articular cartilage during OA as a negative regulator of IL-17 mediated signal transduction. The protective effect of TRAF3 on cartilage is due in part to inhibition of IL-17-induced signaling pathways leading to the production of cartilage catabolic factors. Therefore, TRAF3 is a potential therapeutic target for developing a therapeutic approach to treat OA-induced cartilage degradation.

Claims

1. Use of an active ingredient characterized by:

the active ingredient is selected from:

(a) TNF receptor-related factor 3(TRAF 3);

(b) a precursor capable of being processed in a host to TRAF3 as described in (a);

(c) a polynucleotide capable of being transcribed by a host to form the precursor of (b) and processed to form the TRAF3 of (a);

(d) an expression vector comprising the TRAF3 of (a), or the precursor of (b), or the polynucleotide of (c);

the application of the active ingredients comprises the following steps:

(a) the active ingredient is for inhibiting IL-17 signaling;

2. Use of an active ingredient according to claim 1, characterized in that:

the active ingredient is used for preparing a medicament for treating, preventing, improving or relieving synovitis, cartilage injury or cartilage degeneration caused by arthritis.

3. Use of an active ingredient according to claim 2, characterized in that:

the drug is in a dosage form of gastrointestinal administration or a dosage form of parenteral administration.

4. Use of an active ingredient according to claim 2, characterized in that:

the gastrointestinal administration dosage form is selected from powder, tablet, granule, capsule, solution, emulsion and suspension;

the dosage form of the parenteral administration route is selected from an injection dosage form, a respiratory tract dosage form, a nasal drop, a skin dosage form, a mucous membrane dosage form or a cavity and tract dosage form.

5. Use of an active ingredient according to claim 1, characterized in that:

the active ingredients are also useful for interfering with the binding of IL-17R to Act 1.

6. Use of an active ingredient according to claim 1, characterized in that:

the active ingredients are also useful for inhibiting IL-17 mediated NF- κ B and MAPK pathways, reducing the production of IL-6 and matrix metalloproteinases.

7. Use of an active ingredient according to claim 1, characterized in that:

the active ingredients are also useful for inhibiting IL-17 mediated phosphorylation of p65, p38, JNK and ERK.

8. Use of an active ingredient according to claim 1, characterized in that:

the active ingredients are also useful for inhibiting IL-17 induced MMP-13 production.

9. An arthritis animal model, characterized in that it is selected from one or several of the following animal models:

(B) a DMM-induced rodent comprising a modified, animal model of osteoarthritis;

(C) a rodent comprising a modified animal for use in the construction of an age-related animal model of idiopathic osteoarthritis.

10. An animal model of ulcerative colitis according to claim 9, wherein:

the rodents mentioned in (B) or (C) are also induced by IL-17.