CN114452286A - RAL inhibitors for the prevention and treatment of osteoarthritis - Google Patents

Abstract

The invention provides an application of an active factor, which is characterized in that: the active factors are selected from one or a mixture of a plurality of substances shown as A-D: RBC 8; isomers of RBC 8; derivatives of RBC 8; one or more of stereoisomers, geometric isomers, tautomers, racemates, hydrates, solvates, metabolites and pharmaceutically acceptable salts or prodrugs of RBC 8; the use comprises at least one of the following uses: used for preparing medicines with protective effect on articular cartilage; used for preparing the medicine for delaying cartilage degeneration; can be used for preparing medicine for preventing and treating arthritis (including osteoarthritis, rheumatoid arthritis, etc.).

Description

RAL inhibitors for the prevention and treatment of osteoarthritis

Technical Field

The invention provides an active factor, and particularly relates to an active factor for preventing and treating osteoarthritis.

Background

Osteoarthritis (OA) is the most common joint disease worldwide, with more than 10% of men and 18% of women in people over 60 years of age suffering from it. Data already shows that the social and economic burden caused by OA in developed countries is huge and can account for 1% -2.5% of GDP. With the gradual progress of China into aging society, the prevalence rate of knee osteoarthritis in people over 60 years old in China is 35% -50%, and the prevalence rate is on the rising trend year by year. At present, an effective means for preventing and treating the disease is lacked, and the clinical symptoms can only be relieved by adopting methods of physical therapy, non-steroidal anti-inflammatory drugs (NSAID), joint cavity injection of sodium hyaluronate, glucocorticoid drugs and the like in the early stage of the disease; in the final stage, joint replacement is usually performed, and postoperative complications such as prosthesis loosening and the like are possible, so that heavy economic and psychological burdens are brought to patients and families.

The current research shows that the pathogenesis of OA is complex and is unknown. Research shows that various factors such as abnormal load, aging, trauma and the like of lower limbs can cause synovial inflammation in joints, secretion of inflammatory factors (TNF-a, IL-1 beta, IL-6 and the like), imbalance of joint cartilage homeostasis, release of Matrix Metalloproteinases (MMPs), generation of free radicals and chondrocyte apoptosis, and further cause loss and degeneration of cartilage and subchondral bone, namely imbalance of chondrocyte anabolism. The current clinical drugs for OA mainly include: 1) anti-inflammatory analgesics such as sodium hydrogen peroxide, celecoxib, etc.; 2) drugs that improve osteoarthritic cartilage, such as glucosamine; 3) the knee joint injection medicine, such as sodium hyaluronate and the like, mainly has the functions of relieving inflammation, protecting, lubricating and nourishing cartilage. But for the root cause of OA: the progressive degeneration and disappearance of articular cartilage lacks a targeted control regimen.

At present, OA drugs entering clinical test stage are mainly divided into the directions of targeting bone and cartilage, targeting nerve, targeting immune regulation and the like. Representative drugs for targeted therapy of bone and cartilage are Bisphosphonates (Bisphosphatates), Strontium Ranelate (Strontium Ranelate), recombinant human fibroblast growth factor 18(rhFGF-18, Spriformin), Wnt pathway inhibitors (Loreivivant, cathepsin K inhibitor (MIV711), etc., all of which can pass through but not pass throughThe same mechanism promotes cartilage matrix synthesis and prevents OA progression. The nerve-targeted drugs mainly comprise capsaicin (CNTX-4975) for intra-articular injection, monoclonal antibody (Tanezumab, Fasinumab) for anti-nerve growth factor and the like, and can relieve symptoms of patients by relieving pain. The medicines for targeting immune regulation are Hydroxychloroquine (Hydroxychloroquine), tumor necrosis factor inhibitor (Adalilimumab, Etanercept) and interleukin 1 alpha/beta inhibitor (Anakinra, Lutikizumab)^]Methotrexate (Methotrexate), etc., which alleviate inflammatory responses through immunomodulation to treat OA. The curative effect of the above drugs in clinical experiments is yet to be proved. In the research of preventing and treating OA, a new preventing and treating target point of OA is found, medicine development is carried out aiming at the target point, accurate prevention and treatment are carried out aiming at an OA mechanism, and the regulation of anabolism and catabolism of chondrocytes is the direction of preventing and treating OA.

Disclosure of Invention

In the research of the invention, after protein mass spectrometry analysis and clinical cartilage tissue specimen verification, RAL is found to be obviously highly expressed in OA cartilage tissue, and in the process of OA occurrence and development, RAL plays a negative regulation role, and can obviously promote anabolism of chondrocytes and inhibit catabolism after the expression of RAL is inhibited. In this regard, RAL is thought to play an important role in the development of OA and, by inhibiting its expression, to positively protect chondrocyte metabolism. The RAL inhibitors (inhibitors) can be used as innovative drugs for the prevention and treatment of OA.

Based on the above findings, the present invention provides a novel therapeutic factor or drug against OA disorders. The method comprises the following specific steps:

the invention provides an application of an active factor, which is characterized in that:

the active factors are selected from one or more of the following substances A-D:

RBC8；

isomers of RBC 8;

derivatives of RBC 8;

one or more of stereoisomers, geometric isomers, tautomers, racemates, hydrates, solvates, metabolites and pharmaceutically acceptable salts or prodrugs of RBC 8;

the above uses include at least one of the following uses:

used for preparing medicines with protective effect on articular cartilage;

is used for preparing the medicine for delaying the cartilage degeneration.

In addition, the invention also discloses the application of the active factor, which is characterized in that:

said derivative of RBC8 is selected from BQU57 or an analogue thereof.

In addition, the invention also discloses the application of the active factor, which is characterized in that:

the active factors are selected from one or more of RAL inhibitors;

the above uses include at least one of the following uses:

used for preparing medicines with protective effect on articular cartilage;

is used for preparing the medicine for delaying the cartilage degeneration.

Further, the present invention provides a use of the above active factor, which is characterized in that:

the active factors are also used for preparing medicines for reducing the release of the inflammatory factors of the chondrocytes.

Further, the present invention provides a use of the above active factor, which is characterized in that:

the active factor is also used for preparing medicines for preventing the degradation of cartilage extracellular matrix.

Further, the present invention provides a use of the above active factor, which is characterized in that:

the active factors are also used for preparing medicines for inhibiting cartilage tissue catabolism in an inflammatory state and promoting anabolism.

Further, the present invention provides a use of the above active factor, which is characterized in that:

the above active factors are also useful for the preparation of medicaments or compositions capable of binding to the RAL-GDP complex.

Further, the present invention provides a use of the above active factor, which is characterized in that:

the above active factors are also useful as drugs or ingredients for inhibiting the binding of Ral to its effector RALBP 1.

Further, the present invention provides a use of the above active factor, which is characterized in that:

the above active factor can be used for preparing medicine for promoting cartilage anabolism (such as promoting expression of cartilage anabolism markers ACAN, type II collagen, etc.).

Further, the present invention provides a use of the above active factor, which is characterized in that:

the above active factors can also be used for preparing medicines for inhibiting matrix metalloproteinases (MMPs, such as MMP-3 and MMP-13).

Further, the present invention provides a use of the above active factor, which is characterized in that:

the active factor is also used for preparing medicaments for inhibiting the activation of NF-kB channels.

Further, the present invention provides a use of the above active factor, which is characterized in that:

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

The invention has the following functions and effects:

RAS is one of 5 subfamilies of the RAS superfamily, which contains about 150 gtpases and exerts its molecular biological effects by binding to and hydrolyzing GTP. RAL is named as RAS like (RAL) protein because of the existence of highly similar coding sequence with RAS, while RAL exists in two subtypes, RAL and RALB, both have almost the same coding sequence, perform similar biological functions, and only have difference in C-terminal hypervariable region in three-dimensional structure. RAL is mainly distributed on cell membranes as GTP enzyme, also has the function of combining and hydrolyzing GTP, when the concentration of GTP in cells is 10 times greater than that of GDP, RAL is combined with GTP to be activated under the assistance of RAL guanine nucleotide exchange factors (RALGEF), the activated RAL-GTP complex is combined with a receptor RALBP1 to start a downstream signal path, and then is inactivated into the RAL-GDP complex under the catalysis of RAL GTPase activating proteins (RALGP). In short, when RAL binds to GTP in an activated state and GDP in an inactivated state, it acts as a "molecular switch" to regulate downstream pathways.

RALB can form a complex with secretor 5(SEC5) to directly recruit and activate the atypical ikb family member TBK1, while in the course of primary immunization, activation of TBK1 can activate the NF- κ B pathway, leading to the transcription factor P65 entering the nucleus to initiate downstream transcription and translation, and synthesize Secretory cytokines.

NF-kB is a classical inflammation pathway, and in a non-activated state, the NF-kB protein is in a complex form consisting of two subunits of p65 and p50, is mainly in cells, and forms an inactive trimer with an inhibitor I kB in a non-covalent binding mode. Activation of I κ B kinase (IKK) under upstream stimulation leads to degradation of I κ B, causing p65 to dissociate and phosphorylate, entering the nucleus and functioning as a transcription factor. The expression of inflammatory factors IL-1 beta, TNF-a and IL-6 is caused by the activation of the pathway in OA tissues, and meanwhile, the secretion of the inflammatory factors further activates an NF-kB pathway to form a circulation amplification process, and finally, the balance of synthesis and catabolism of chondrocytes is destroyed, so that OA occurs in the cartilage tissues.

Based on the above theoretical considerations, the present inventors have attempted to use specific inhibitors of RAL, which have been found to inhibit coupling to the receptor RALBP1 by binding to the RAL-GDP complex, and thus inhibit the physiological effects of RAL. The experiment carried out by synchronously using RBC8 (a specific inhibitor of RAL) shows that IL-1 beta induces a mouse primary chondrocyte OA model and intervenes by using RBC8, and the result shows that the inhibition of RAL can promote the expression of a cartilage anabolism marker ACAN and inhibit the synthesis of MMP-3 at a certain concentration. The RAL inhibitor RBC8 is proved to have obvious articular cartilage protection effect.

Therefore, the invention provides that RAL has high expression in OA degenerative cartilage and plays an important role in the occurrence and development of OA, and the inhibition of the expression of RAL can reduce the release of chondrocyte inflammatory factors by inhibiting the activation of downstream important signals such as NF-kB channels, prevent the degradation of chondrocyte extracellular matrix and further delay the cartilage degeneration.

Drawings

FIG. 1 GDP-GTP cycling of RAL GTPase.

FIG. 2 RAL is highly expressed in human OA degenerated cartilage tissue species.

Wherein (A) safranin-fast green, Alnew blue detection shows that the OA group joint cartilage is degenerated and the extracellular matrix content is reduced compared with the normal control; the immunohistochemical staining test shows that RAL is highly expressed in the articular cartilage of the OA group (black arrows indicate positive detection reaction) compared with a normal control, and the immunofluorescence test also shows that RAL is highly expressed in articular cartilage cells of the OA group (green is positive expression);

(B) western blot shows that when detecting that the OA pathogenic important inflammatory factor IL-1 beta is 10ng/ml and 20ng/ml, the high-expression RAL of the chondrocytes can be stimulated;

(C) western blot detection shows that IL-1 beta of 10ng/ml can stimulate RAL high expression when stimulating chondrocytes for more than 12 hours;

(D) real-time fluorescent quantitative PCR detection shows that RAL is highly expressed in the articular cartilage of OA group compared with normal control. ". indicates p < 0.001.

Fig. 3 RAL inhibitor RBC8 inhibits cartilage degeneration.

Wherein, (A) RBC8 molecular structural formula;

(B) detecting the effect of different concentrations of RBC8 on chondrocyte activity;

(C) western blot detection of the influence of RBC8 on the expression of articular chondrocyte Col2, ACAN and MMP13 proteins shows that RBC8 obviously saves the inhibition effect of OA pathogenic important inflammatory factor IL-1 beta on Col2 and ACAN, promotes the expression of cartilage anabolism factors Col2 and ACAN under OA pathological conditions, and inhibits the expression of cartilage catabolism factor matrix metalloproteinase MMP-13 under OA pathological conditions.

(D) Immunofluorescence results show that RBC8 promotes the expression of articular chondrocyte anabolic factor Col2 under OA pathological conditions.

(E) Immunofluorescence results show that RBC8 promotes the expression of the articular chondrocyte anabolic factor ACAN under OA pathological conditions.

(F) Immunofluorescence results show that RBC8 inhibits the expression of articular chondrocyte catabolic factor MMP13 under OA pathological conditions.

FIG. 4. cartilage tissue mass culture showed that RBC8 significantly inhibited articular cartilage degeneration.

FIG. 5 BQU57 is used for preventing and treating cartilage degeneration and protecting articular cartilage.

(A-B) Western blot results.

(C) And (4) performing immunofluorescence.

(D) And (4) performing immunofluorescence.

Detailed Description

Experimental method for verifying RAL expression condition in degenerative cartilage tissue and cells

(1) Collecting articular cartilage of a patient undergoing total knee joint replacement for knee osteoarthritis in clinic, and classifying the articular cartilage into a normal group and an OA group according to whether cartilage tissues are damaged or not and whether the cartilage tissues are smooth or not, wherein the articular cartilage is the OA group if the cartilage surface is uneven and granular and subchondral bone is locally visible; if the cartilage surface is smooth and porcelain white or yellowish, no cartilage damage is caused, and the cartilage is a relatively normal group. Detecting RAL expression condition by Western Blot and immunofluorescence;

wherein,

the specific method for Western Blot is as follows:

1.1 tissue protein extraction

1) Shearing tissue into small pieces

2) Lysate is added at a rate of 200. mu.L RIPA lysate per 20mg of tissue (Shenneng Cao, China), and if high protein concentration is required, the lysate is reduced accordingly.

Note: after lysis of all cell/tissue samples, centrifugation at 12000g for 10min at 4 ℃ and careful transfer of supernatant to new EP tubes, the protein can be stored for a long period in a-80 ℃ freezer.

1.2 protein quantification

1) The protein was quantified using BCA protein detection kit (Invitrogen, USA). Completely dissolving the protein standard, taking a proper amount of 25mg/mL protein standard, and diluting with RIPA lysate to a final concentration of 0.5 mg/mL.

2) According to the number of samples, a proper amount of BCA working solution is prepared by adding 1 volume of BCA reagent A and 1 volume of BCA reagent B50 (1:50) and is fully mixed.

3) Adding standard substance into standard substance well of 96-well plate according to 0, 1, 2, 4, 8, 12, 16, 20 μ l, adding standard substance diluent to make up to 20 μ l, wherein the concentrations of the standard substance are respectively 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/ml; add the appropriate volume of sample to the sample well of a 96-well plate. If the sample is less than 20. mu.l, the standard dilution is added to make up to 20. mu.l.

4) Add 200. mu.l BCA mixture to each well and leave at 37 ℃ for 30 minutes.

5) And measuring the light absorption value of OD562nm by using a microplate reader, and calculating the protein concentration of the sample according to the standard curve and the used sample volume.

1.3 denaturation of proteins

The quantified proteins were blended with SDS PAGE Loading Buffer (Takara, Japan) at a ratio of 4:1 and boiled at 100 ℃ for 5 minutes. The denatured sample can be stored for a long time at minus 80 ℃.

1.4 protein electrophoresis

1) And cleaning the glass plate, airing and placing the glass plate on a glue making frame.

2) Separating gel is prepared according to the instruction of SDS-PAGE gel rapid preparation kit (Yazyme, China), and the specific concentration is determined according to the protein amount.

3) 7ml of separation gel is slowly added into the placed gel making plate along the glass plate, and no air bubbles exist. Then 2ml of absolute ethyl alcohol is added to seal the separation gel, the separation gel is waited for 20 minutes until the separation gel is solidified, and the ethyl alcohol is poured out and sucked dry.

4) Preparing concentrated gel according to the SDS-PAGE gel rapid preparation kit instruction, adding enough concentrated gel along the glass plate again until just falling off, inserting the comb, waiting for about 15 minutes until the concentrated gel can be solidified, and slightly and vertically pulling the comb upwards.

5) After gel preparation, gel and glass plate were placed in an electrophoresis tank, and 1 Xelectrophoresis buffer (Yazyme, China) was added. Based on the protein quantification (about 20. mu.g per well), the loading volume was calculated and the protein sample was slowly added per well along the wall. Wherein 5 μ l of protein molecular weight standard (Biyuntian, China) is added.

6) Then, after electrophoresis at 90V for 30 minutes, electrophoresis at 120V is carried out for 1-2 hours.

1.5 Rotary film sealing

1) After electrophoresis, the gel was rinsed in TBST (Yazyme, China) for 2 minutes, and then soaked in a membrane-transferring solution (Yazyme, China) for 5 minutes.

2) And soaking the sheared PVDF membrane in methanol for 5s, and balancing in a membrane transferring solution for 5 minutes.

3) The membrane transfer composite is placed from the positive electrode to the negative electrode in the order of 'white frame-sponge-filter paper-PVDF membrane-glue-filter paper-sponge-black frame', and air bubbles inside are thoroughly removed.

4) The membrane is transferred by ice bath, the constant current of 300mA and the membrane is transferred for 2 hours at the temperature of 4 ℃, and the protein on the separation gel is transferred to the PVDF membrane.

5) After the membrane transfer is finished, ponceau is dyed for 5 minutes, TBST is rinsed for 3-5 minutes, the process is repeated for 2-3 times, and bands near the target protein are cut off.

6) Blocking with 5% BSA (raw, China) (1X TBST, 5g +100mL) was performed for 1 hour at room temperature.

1.6 antibody incubation

1) After blocking, primary antibody was prepared in blocking solution (5% BSA in 1 XTSST) and the membrane incubated overnight at 4 ℃ in a shaker.

2) After incubation was complete, the 1 × TBST rinse was 5 minutes and repeated 3 times.

3) Fluorescent secondary antibody (Li-cor, USA) was prepared from blocking solution (species selected according to primary antibody) and incubated for 1h at room temperature in a shaker.

4) After incubation was complete, the 1 × TBST rinse was repeated 3 times for 5 minutes.

1.7 protein detection

1) The film was placed into an Odyssey two-color infrared laser imaging system for photography.

The immunofluorescence method is specifically as follows:

1) paraffin sections are dewaxed and hydrated conventionally, xylene is used twice for 10-20 minutes each time, and gradient alcohol is used for 100% -95% -85% -75% each time for 5 minutes.

2) TBST washes were three times for 3 minutes each.

3) And putting the slices into a wet box, absorbing water around the tissues by using a dust-free paper towel, and correspondingly repairing the tissue antigens for 45 minutes according to the requirements of different antibodies.

4) TBST was washed 3 times and enough 3% H2O2 solution (30% H2O2 and methanol as 1: 9, 1ml for 12 sections) at room temperature for 10 minutes to block the activity of endogenous peroxidase in the tissue.

5) TBST was washed three times for 5 minutes each. Moisture was removed from the surrounding tissue and a pen was used to draw a circle around the tissue. 5% BSA blocking at RT for 1h

6) The blocking solution was removed and washed three times for 5 minutes each with TBST. Primary anti-Mmp 13(Abcam,1:200, ab39012), type X collagen (Abcam,1:1000, ab58632), GFP (Abcam,1:1000, ab290)) was diluted with 5% BSA depending on the requirements of the different antibodies. 50 μ l of primary antibody was added to each section overnight at 4 ℃.

7) The primary antibody was removed and washed three times for 5 minutes each with TBST.

8) A fluorescent secondary antibody (abcam,1:1000, USA) was added. Incubate for 1 hour at room temperature in the dark.

9) The secondary antibody was removed and incubated with DAPI staining solution (bosd, china) for 10min at room temperature.

10) Rinse three times with TBST for 5 minutes each at room temperature.

11) The result was mounted with mounting medium (bosd, china) and observed under a fluorescent microscope.

(2) The damage degree of the cartilage is observed by pre-staining safranin fast green, the collected cartilage tissues are graded according to OARSI scores (grades O, 1, 2, 3, 4, 5 and 6), total RNA is respectively extracted from the cartilage with different pathological degrees, and whether the expression level of RAL mRNA is related to the disease progression degree is detected. As the chondrocytes of the human cartilage tissue are sparse and the extracellular matrix components are excessive, the mucopolysaccharide extracted by the traditional Trizol method has more pollution, and the total RNA is difficult to purify. Therefore, the laser microdissection instrument is used for cutting and collecting 50mg of single cells or cell communities and then extracting total RNA;

wherein,

the safranin fast green dyeing method specifically comprises the following steps:

1) human cartilage tissue was embedded, sectioned, deparaffinized to water: xylene I10min and xylene II 10min (dewaxing process, dewaxing is thorough); 100% ethanol I for 5min, and 100% ethanol II for 5 min; 95% ethanol I for 5min, and 95% ethanol II for 5 min; soaking in 85% ethanol for 5 min; soaking in 75% ethanol for 5 min; rinsing with tap water.

2) Flushing with tap water for 30 seconds;

3) fast green (Sigma, Germany) (0.1% fast green solution: weighing 0.5g safranin powder, dissolving with 500ml deionized water) and dyeing for 5 minutes;

4) washing with tap water for 1 minute;

5) safranin (Sigma, Germany) (0.2% safranin solution: weighing 1g of safranin powder, dissolving with 500ml of deionized water) and dyeing for 5 minutes;

6) 1% glacial acetic acid was color separated for 1 second;

7) dehydrating with 95% ethanol for 1 min for 2 times;

8) dehydrating with 100% ethanol for 2 min, 2 times;

9) xylene was clear for 5 minutes, 2 times;

10) and (5) sealing by using neutral gum.

Regarding the OARSI scoring criteria:

level 0: normal cartilage;

level 0.5: loss of safranin O staining without tissue structural changes;

level 1: the surface layer without cartilage loss has micro fibrosis;

and 2, stage: the vertical crack is downward to the surface cartilage, and part of the surface cartilage is lost;

and 3, level: vertical fissures/defects reach calcified cartilage affecting the articular surface < 25%;

4, level: the vertical fissures/defects reach the calcified cartilage, affecting the articular surface 25-50%;

and 5, stage: the vertical fissures/defects reach the calcified cartilage, affecting the articular surface 50-75%;

stage 6: vertical fissures/defects reach calcified cartilage affecting the articular surface > 75%

7(3) isolation and culture of primary chondrocytes of mice: taking a newborn mouse born within 3 days, carrying out anesthesia over chloral hydrate until death, soaking the newborn mouse in 75% ethanol for 15min for disinfection, taking two lower limbs of the newborn mouse, carefully separating and removing muscle ligaments around a knee joint, removing pink femur and tibia, only keeping transparent cartilage of the knee joint such as millet grain size, cutting into muddy flesh, soaking the minced flesh in 0.2% type II collagenase at 37 ℃, carrying out shake digestion for 4-6h until tissues disappear or a culture solution is obviously turbid, stopping digestion, centrifuging and re-suspending, inoculating the minced flesh into a culture dish, and transferring the minced flesh to P1-P2 for use. Inducing a primary OA chondrocyte model of a mouse through IL-1 beta with different concentration gradients, and detecting whether RAL is highly expressed or not and whether the RAL is related to the degeneration degree or not;

the grouping is as follows:

NC group;

② IL-1 beta group (5 ng/ml);

③ IL-1 beta group (10 ng/ml);

IL-1 beta group (20 ng/ml);

experimental method for verifying influence of RAL inhibition/overexpression on chondrocyte metabolism and cartilage tissue extracellular matrix

2-1. in vitro experiment:

(1) constructing a RAL knockdown siRNA transfected mouse ATDC5 cell line, and detecting the anabolism index of the chondrocyte by Western Blot: col2a1, ACAN, SOX9 and catabolic indices: MMP-3, MMP-13 and ADAMTS5, and collecting culture medium to perform ELISA to detect the secretion of IL-1 beta, TNF-a and IL-6 of chondrocytes;

(2) constructing a RAL overexpression plasmid transfected mouse ATDC5 cell line, and detecting the anabolism index of the chondrocyte by Western Blot: col2a1, ACAN, SOX9 and catabolic indices: MMP-3, MMP-13 and ADAMTS5, and collecting culture medium to perform ELISA to detect the secretion of IL-1 beta, TNF-a and IL-6 of chondrocytes;

(3) tissue block culture: aseptically separating mouse femoral head cartilage, respectively culturing tissue blocks (as shown in figure 4), changing culture solution for 2-3 days, simultaneously adding IL-1 beta to induce cartilage endochondral cell degeneration, continuously acting for 2 weeks, and adding RAL inhibitor RBC8 to intervene for 2 weeks on the basis of IL-1 beta induction in treatment group. Tissue blocks were collected after 2 weeks for pathological analysis: the safranin fast green staining and the alcian blue staining are used for detecting the change of the cartilage extracellular matrix, and the immunohistochemistry is used for detecting the anabolism indexes of cartilage histiocytes and the extracellular matrix: col2a1, ACAN, SOX9 and catabolic indices: expression of MMP-3, MMP-13, ADAMTS 5;

the grouping is as follows:

NC group;

IL-1 beta group;

③ IL-1 beta + RBC8 group

Wherein,

the method for constructing the RAL knockdown siRNA/overexpression plasmid transfected mouse ATDC5 cell line is as follows:

1) transfecting ATDC5 cell line with 70-90% confluency;

2) lipo3000 reagent (Thermofisiher, USA)3.75ul was diluted with Opti-MEM serum free medium (Thermofisiher, USA)125ul and mixed well (two tubes);

3) 5ug of DNA (Jima gene, China) was diluted with 250ul of Opti-MEM serum-free medium to prepare a premixed DNA solution (P300010 ul was added if over-expression plasmid was transfected), and the solution was mixed well;

4) diluted DNA (1: 1 proportion), and incubating for 10-15min at room temperature;

5) adding the incubated DNA-lipid complex to cells;

6) after incubation of the cells at 37 ℃ for 2-4 days, the transfected cells were analyzed for downstream detection.

Regarding the ELISA kit:

1) the number of pre-coated strips required for one experiment was calculated and determined according to the ELISA kit (bi yun, china) procedure, and the required strips were taken out and placed in a 96-well frame.

2) Preparing a standard substance and drawing a standard curve, and meanwhile, arranging a background-corrected hole, wherein only TMB solution and stop solution are added into the hole.

3) Samples or standards with different concentrations are added into corresponding wells according to 100 mul/well, reaction wells are sealed by sealing plates, and the wells are incubated for 120 minutes at room temperature.

4) The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

5) Biotinylated antibody 100. mu.l/well was added, the reaction wells were sealed with a sealing plate and incubated at room temperature for 60 minutes.

6) The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

7) 100. mu.l/well of Streptavidin was labeled with horseradish peroxidase. The reaction wells were sealed with a sealing plate and incubated at room temperature in the dark for 20 minutes.

8) The plate was washed 5 times and the last time was patted dry on thick absorbent paper.

9) Adding 100 mul/hole of color reagent TMB solution, sealing the reaction hole with sealing plate, and incubating for 20min at room temperature in dark.

10) Add stop solution 50. mu.l/well and measure A450 value immediately after mixing.

(4) Intervention experiment of BQU57 (see FIG. 5)

Cell culture and drug intervention

3) Inoculating ATDC5 cells in a six-well plate;

4) BQU (0.1uM,1uM) pretreatment for 24 hours, followed by IL-1 β (20ng/ml) intervention for 12 hours;

5) the protein was collected for subsequent studies.

Western Blot detection

2.1 protein extraction

1) Washing adherent cells three times with pre-cooled PBS;

2) lysate is added at a rate of 100 μ L lysate per well, and if high concentration protein is required, lysate can be reduced accordingly.

Note: after lysis of all cell samples, centrifugation at 12000g for 10min at 4 ℃ carefully transferred the supernatant to a new EP tube and the protein could be stored for a long period in a-80 ℃ freezer.

2.2 protein quantification

6) Protein was quantified using the BCA protein assay kit. Completely dissolving the protein standard, taking a proper amount of 25mg/mL protein standard, and diluting with RIPA lysate to a final concentration of 0.5 mg/mL.

7) According to the number of samples, a proper amount of BCA working solution is prepared by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and the mixture is fully mixed.

8) Adding standard substance into standard substance well of 96-well plate according to 0, 1, 2, 4, 8, 12, 16, 20 μ l, adding standard substance diluent to make up to 20 μ l, wherein the concentrations of the standard substance are respectively 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/ml; add the appropriate volume of sample to the sample well of a 96-well plate. If the sample is less than 20. mu.l, the standard dilution is added to make up to 20. mu.l.

9) Add 200. mu.l BCA mixture to each well and leave at 37 ℃ for 30 minutes.

10) And measuring the light absorption value of OD562nm by using a microplate reader, and calculating the protein concentration of the sample according to the standard curve and the used sample volume.

2.3 denaturation of proteins

The quantified protein and SDS PAGE Loading Buffer are mixed according to the proportion of 4:1 and boiled for 5 minutes at 100 ℃. The denatured sample can be stored for a long time at minus 80 ℃.

2.4 protein electrophoresis

7) And cleaning the glass plate, airing and placing the glass plate on a glue making frame.

8) Firstly, separating gel is prepared according to the instruction of the SDS-PAGE gel rapid preparation kit, and the specific concentration is determined according to the protein amount.

9) 7ml of separation gel is slowly added into the placed gel making plate along the glass plate, and no air bubbles exist. Then 2ml of absolute ethyl alcohol is added to seal the separation gel, the separation gel is waited for 20 minutes until the separation gel is solidified, and the ethyl alcohol is poured out and sucked dry.

10) The gels were prepared according to the SDS-PAGE gel rapid formulation kit instructions (petyuntian, P0012AC), a sufficient amount of gel was added to the glass plate again until just after the roof fall, the comb was inserted, the gel allowed to set after about 15 minutes, and the comb was pulled gently upright.

11) After gel preparation, gel and glass plates were placed in an electrophoresis tank and 1 Xelectrophoresis buffer was added. Based on the protein quantification (about 20. mu.g per well), the loading volume was calculated and the protein sample was slowly added per well along the wall. Wherein 5 μ l of protein molecular weight standard (Biyunyan, P0066) was added.

12) Then, after electrophoresis at 90V for 30 minutes, electrophoresis at 120V is carried out for 1-2 hours.

2.5 Rotary film sealing

7) After electrophoresis, the gel needs to be rinsed in TBST for 2 minutes, and then soaked in the membrane transferring solution for 5 minutes.

8) And soaking the sheared PVDF membrane in methanol for 5s, and balancing in a membrane transferring solution for 5 minutes.

9) The membrane transfer composite is placed from the positive electrode to the negative electrode in the order of 'white frame-sponge-filter paper-PVDF membrane-glue-filter paper-sponge-black frame', and air bubbles inside are thoroughly removed.

10) The membrane is transferred by ice bath, the constant current is 300mA, the membrane is transferred for 2 hours at the temperature of 4 ℃, and the protein on the separation gel is transferred to the PVDF membrane.

11) After the membrane transfer is finished, ponceau is dyed for 5 minutes, TBST is rinsed for 3-5 minutes, the process is repeated for 2-3 times, and bands near the target protein are cut off.

12) Blocking was performed with 5% BSA (1X TBST, 5g +100mL) for 1 hour at room temperature.

2.6 antibody incubation

5) After blocking, primary antibody was prepared in blocking solution (5% BSA in 1 XTSST) and the membrane incubated overnight at 4 ℃ in a shaker.

6) After incubation was complete, the 1 × TBST rinse was repeated 3 times for 5 minutes.

7) Fluorescent secondary antibodies (selected according to the primary antibody) were prepared in blocking solution and incubated for 1h in shaker at room temperature.

8) After incubation was complete, the 1 × TBST rinse was repeated 3 times for 5 minutes.

2.7 protein detection

2) According to BeyoECL Plus, a proper amount of solution A and solution B with equal volume are taken and mixed into a working solution.

3) The filter paper aspirates the residual wash solution without encountering a protein band.

4) Dripping the working solution on the transfer printing film till the working solution is completely covered, and standing for 1-2 minutes at room temperature

5) And (4) taking the membrane, discarding the BeyoECL Plus working solution, and slightly absorbing excessive liquid by using absorbent paper. The film is placed between two sheets of preservative film, and the film is placed in a chemiluminescence imager for photographing.

Cellular immunofluorescence

1) Inoculating cells on a cell slide;

2) soaking the cell-crawled slide in PBS 3 times for 3min each time in the culture plate;

3) fixing the slide with 4% paraformaldehyde for 15min, and washing the slide with PBS for 3 times, each for 3 min;

4) preparing 0.5% Triton X-100(PBS) and allowing to permeate at room temperature for 20 min;

5) soaking and washing the slide with PBS for 3 times, each time for 3min, sucking dry PBS with absorbent paper, dripping normal goat serum on the slide, and sealing at room temperature for 30 min;

6) absorbing sealing liquid by absorbent paper, not washing, dripping enough diluted primary antibody on each glass slide, putting the glass slide into a wet box, and incubating overnight at 4 ℃;

7) adding a fluorescent secondary antibody: soaking and washing the climbing sheet with PBST for 3 times, each time for 3min, sucking the excess liquid on the climbing sheet with absorbent paper, dripping diluted fluorescent secondary antibody, incubating in a wet box at 20-37 deg.C for 1h, soaking and washing the climbing sheet with PBST for 3 times, each time for 3 min;

note that: all subsequent processing steps were performed as dark as possible from the addition of the fluorescent secondary antibody.

8) Counterstaining the nucleus: dripping DAPI, incubating for 5min in dark, staining the specimen for nucleus, and washing off excessive DAPI 5min × 4 times by PBST;

9) and (3) absorbing the liquid on the slide by using absorbent paper, sealing the slide by using sealing liquid containing an anti-fluorescence quenching agent, and observing and acquiring an image under a fluorescence microscope.

Third, results and analysis

High expression of RAL protein in OA degenerated cartilage

Collecting articular cartilage of a patient who is subjected to total knee replacement due to knee osteoarthritis, dividing the articular cartilage into a Normal group and an OA group according to the completeness of articular surfaces, firstly performing safranin-fast green staining, wherein the staining result shows that the thickness of an articular cartilage layer of the OA group is remarkably thinner than that of the articular cartilage layer of the Normal group, the alcian blue staining result shows that the content of OA group proteoglycan is remarkably smaller than that of the Normal group, immunohistochemical staining shows that the expression of RAL of the OA group is remarkably lower than that of the Normal group, separating chondrocytes from the Normal and OA groups, and detecting the expression of RAL by immunofluorescence, wherein the expression of RAL in the chondrocytes from the OA group is remarkably higher than that of the chondrocytes from the Normal group (figure 2A). The chondrocytes were stimulated with different concentrations of IL-1(0ng/ml,5ng/ml,10ng/m and 20mg/ml) for 24 hours, and the expression of RAL protein was detected by Western blot, showing that IL-1 promotes the expression of RAL protein in chondrocytes and is concentration-dependent (FIG. 2B). The expression of RAL was detected by western blot using 10ng/ml IL-1 stimulation of chondrocytes for 0 hr, 6 hr, 12 hr, 24 hr, 36 hr and 48 hr, and the results showed that the expression of RAL protein increased with increasing stimulation time, with time dependence (fig. 2C). The real-time fluorescent quantitative PCR detection of RAL mRNA expression in articular cartilage of both Normal group and OA group showed that RAL mRNA expression in articular cartilage of OA group was significantly higher than that in articular cartilage of Normal group (FIG. 2D).

Wherein,

the specific method for Real-time PCR analysis is as follows:

1. extraction of tissue total RNA

1) Cells and cell clusters in different grades of articular cartilage tissues were cut and collected by using a microdissection instrument (Leica, Germany).

2) The paraffin tissue collected was subjected to total RNA extraction as specified in the paraffin tissue total RNA extraction kit (Qiagen, Germany).

3) After the sample was dissolved in RNase-free water (20-50. mu.l), the concentration and purity of RNA were measured by Nanodrop 2000 (Thermofoisher, USA) and OD260/OD280 should be between 1.8 and 2.0. RNA samples were stored at-80 ℃ or directly subjected to RT-PCR.

4) All the articles used in this test need to be DEPC treated.

Reverse transcription reaction of RNA:

1) preparing reverse transcription reaction mixed solution on ice according to the following proportion:

2) after the reaction mixture is mixed evenly, reverse transcription is carried out according to the following parameters:

3) the sample was stored at-20 ℃ for use.

3.Real-time PCR

Detection of gene mRNA expression: the takara Real-time PCR detection reagent (CAT: RR420A) was used.

The primer sequences are detailed in the table below.

1) A Real-time PCR reaction solution was prepared on ice in the following composition, and the reaction was carefully carried out in the dark.

2) And (3) centrifuging for a short time, throwing the solution to the bottom of the tube, wherein the reaction parameters are as follows:

3) after the reaction is finished, determining the amplification curve and the dissolution curve of Real-time PCR. GAPDH was used as an internal reference gene, and PCR was performed by the synthetic CT method.

RAL inhibitor RBC8 inhibits IL-1 induced degradation of articular cartilage extracellular matrix

RBC8 is an inhibitor of RAL, has a molecular structure shown in FIG. 3A, and stimulates chondrocytes using RBC8 at different concentrations (0. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M and 40. mu.M), and shows that RBC8 at 1. mu.M and 5. mu.M had no effect on chondrocyte activity (FIG. 3B). Western blot results show that IL-1 can inhibit the expression of Col2 and ACAN proteins and can promote the expression of MMP-13 protein, and RBC8 can promote the expression of Col2 and ACAN proteins and can inhibit the expression of MMP-13 protein (figure 3C). The real-time fluorescent quantitative PCR detection result shows that IL-1 can inhibit the expression of Col2 and ACAN mRNA and can promote the expression of MMP-13mRNA, while RBC8 can promote the expression of Col2 and ACAN mRNA and can inhibit the expression of MMP-13mRNA (FIG. 3D). Immunofluorescence detection results show that IL-1 can inhibit the expression of Col2 and ACAN proteins and can promote the expression of MMP-13 protein, while RBC8 can promote the expression of Col2 and ACAN proteins and can inhibit the expression of MMP-13 protein (FIG. 3E).

The specific method for alcian blue staining is as follows:

1) paraffin sections are dewaxed into water conventionally;

2) washing with tap water for 30 seconds;

3) alcian blue (Sigma, Germany) was stained for 30 minutes (1 g alcian blue powder was weighed, dissolved in 100ml of 3% glacial acetic acid, and the pH was adjusted to 2.5 with acetic acid);

4) washing with tap water for 2 minutes;

5) dehydrating with 95% ethanol for 3min for 1 time;

6) dehydrating with 100% ethanol for 3min for 2 times;

7) xylene was clear for 5 minutes, 2 times;

8) sealing with neutral gum;

specific methods for immunohistochemical staining are as follows:

1) paraffin sections are dewaxed and hydrated conventionally, xylene is used twice for 10-20 minutes each time, and gradient alcohol is used for 100% -95% -85% -75% each time for 5 minutes.

2) TBST (TBS added Tween-20 to a final concentration of 0.05%) was washed three times for 3 minutes each. Or directly rinsing with DD water for several times (changing the jar).

3) The sections were placed in a wet box and the moisture around the tissue was removed with a dust free paper towel and the tissue antigens were repaired for 45 minutes with pepsin according to the requirements of the different antibodies.

4) TBST was washed 3 times and enough 3% H2O2 solution (30% H2O2 and methanol as 1: 9, 1ml for 12 sections) at room temperature for 10 minutes to block the activity of endogenous peroxidase in the tissue.

5) TBST was washed three times for 5 minutes each. Moisture was removed from the surrounding tissue and a pen was used to draw a circle around the tissue. 5% BSA (0.5g BSA (4 ℃ refrigerator) +10ml TBST) blocked for 1h at RT

6) The blocking solution was removed and washed three times for 5 minutes each with TBST. Primary anti-Mmp 13(Abcam,1:200, ab39012), type X collagen (Abcam,1:1000, ab58632), GFP (Abcam,1:1000, ab290)) was diluted with 5% BSA depending on the requirements of the different antibodies. 50 μ l of primary antibody was added to each section overnight at 4 ℃.

7) Remove primary antibody, wash three times for 5 minutes each with TBST.

8) Adding secondary antibody (Mixin, China). HRP labeled IgG. Incubate at 37 ℃ for one hour in an incubator.

9) Secondary antibody was removed and washed three times for 5 minutes each with TBST. 50 μ l of freshly prepared DAB solution was added to each section and left at room temperature for 3-10 minutes. The DAB solution is ready for use. When the brown color appears quickly during the observation, the staining should be stopped early, and the observation can be carried out under a microscope if the brown color is uncertain.

10) Excess DAB solution was removed, slightly rinsed with tap water, 3 minutes with hematoxylin counterstain, 2 minutes with tap water and turned blue.

11) The 95% ethanol is dehydrated for 5 minutes 2 times.

12) 100% ethanol was dehydrated for 5 minutes, 2 times.

13) Xylene was clear for 5 minutes, 2 times.

14) And (5) sealing by using neutral gum.

RAL inhibitor RBC8 inhibits cartilage tissue mass catabolism and promotes anabolism in inflammatory states

To test the effect of RBC8 on articular cartilage catabolism and anabolism, cartilage tissue blocks were cultured in vitro, IL-1 β was added to mimic the inflammatory state of articular cartilage, RBC8 was added, and the effect of RBC8 on cartilage tissue block metabolism was tested. As shown in FIG. 4, the results of safranin-fast green staining and alcian blue staining showed that IL-1 β can promote the degradation of matrix in the tissue mass, while RBC8 can inhibit the degradation of matrix in the tissue mass caused by IL-1 β. Immunohistochemical staining results show that IL-1 beta can inhibit the expression of ACAN and Col2 in cartilage tissue masses, and RBC8 can slow down the decrease of ACAN and Col2 expression caused by IL-1 beta. Immunohistochemical staining results show that IL-1 beta, an important inflammatory factor for OA, can promote the expression of MMP-13 in cartilage tissue masses, and RBC8 can slow down IL-1 beta to cause the reduction of MMP-13 expression.

Thus, it was shown by the cartilage tissue mass culture that RBC8 can significantly inhibit articular cartilage degeneration and protect the cartilage matrix. Obviously saves the inhibition effect of IL-1 beta on Col2 and ACAN which are important inflammation factors for pathogenic OA, promotes the expression of Col2 and ACAN which are cartilage anabolism factors under the pathological condition of OA, and inhibits the expression of matrix metalloproteinase MMP-13 which is cartilage catabolism factors under the pathological condition of OA.

BQU57 can be used for preventing and treating cartilage degeneration and protecting articular cartilage.

As shown in the Western blot results of FIGS. 5A and 5B, BQU57 obviously rescues the inhibition effect of IL-1 beta on Col2, promotes the expression of cartilage anabolism factor Col2 under the OA pathological condition, and inhibits the expression of cartilage catabolism factor matrix metalloproteinase MMP-13 under the OA pathological condition. And B is quantitative analysis of the A picture.

As shown by immunofluorescence results in FIG. 5C, BQU57 promotes the expression of the articular chondrocyte anabolic factor Col2 under OA pathological conditions.

As shown in the immunofluorescence results of FIG. 5D, BQU57 inhibited the expression of the articular chondrocyte catabolic factor MMP-13 under pathological conditions of OA.

Claims (10)

1. Use of an active factor, characterized by:

the active factors are selected from one or a mixture of a plurality of substances shown as A-D:

A.RBC8；

isomers of rbc 8;

a derivative of rbc 8;

one or more of stereoisomers, geometric isomers, tautomers, racemates, hydrates, solvates, metabolites and pharmaceutically acceptable salts or prodrugs of RBC 8;

the use comprises at least one of the following uses:

I. used for preparing medicines with protective effect on articular cartilage;

II, preparing the medicine for delaying cartilage degeneration.

2. The use of an active factor according to claim 1, wherein:

said derivative of RBC8 is selected from BQU57 or an analogue thereof.

3. Use of an active factor, characterized by:

the active factor is selected from one or a mixture of more of RAL inhibitors;

the use comprises at least one of the following uses:

III, preparing a medicament with a protective effect on articular cartilage;

IV, the preparation of the medicine for delaying cartilage degeneration.

4. Use of an active factor according to any of claims 1 to 3, wherein:

the active factor is also used for preparing medicaments for reducing the release of the inflammatory factors of the chondrocytes;

or

The active factor is also used for preparing medicaments for preventing the degradation of cartilage extracellular matrix.

5. Use of an active factor according to any of claims 1 to 3, wherein:

the active factor is also used for preparing a medicament for inhibiting the catabolism of cartilage tissues in an inflammatory state and promoting anabolism.

6. Use of an active factor according to any of claims 1 to 3, wherein:

the active factor is also used for preparing medicaments or components capable of being combined with the RAL-GDP complex.

7. Use of an active factor according to any of claims 1 to 3, wherein:

the active factor is also useful as a drug or ingredient that inhibits the binding of Ral to its effector RALBP 1.

8. Use of an active factor according to any of claims 1 to 3, wherein:

the active factor is also used for preparing medicaments for promoting cartilage anabolism.

9. Use of an active factor according to any of claims 1 to 3, wherein:

the active factor is also used for preparing medicaments for inhibiting matrix metalloproteinase.

10. Use of an active factor according to any of claims 1 to 3, wherein:

the active factor is also used for preparing medicaments for inhibiting the activation of NF-kB channels.

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