阅读说明：本技术 川陈皮素的一种新用途及其组合物 (New application of nobiletin and composition thereof ) 是由 刘元艳 刘蕊 宋钰蓉 李晨曦 华正来 罗欣怡 李阳 于 2021-07-16 设计创作，主要内容包括：本发明属于药物制备技术领域,具体涉及川陈皮素的一种新用途及其组合物。其应用于制备降低关节炎滑膜组织中P-gp表达的药物。川陈皮素和对甲氨蝶呤联用可以减少软骨和骨骼损伤,并改善肿胀和关节炎损伤。另一方面,川陈皮素可以逆转该关节炎损伤者的P-gp表达,从而减少对甲氨蝶呤的流出并维持对甲氨蝶呤的功效。川陈皮素和对甲氨蝶呤之间有明显的协同作用,在川陈皮素的非细胞毒性浓度下,川陈皮素+对甲氨蝶呤共同处理可恢复RA-FLS/对甲氨蝶呤细胞的生长抑制和细胞凋亡。川陈皮素可以作为潜在化学增敏剂克服RA中的耐药性。(The invention belongs to the technical field of medicine preparation, and particularly relates to a new application of nobiletin and a composition thereof. It is applied to preparing the medicine for reducing the expression of P-gp in the synovial tissue of arthritis. The combination of nobiletin and methotrexate reduces cartilage and bone damage and ameliorates swelling and arthritic damage. On the other hand, nobiletin can reverse P-gp expression in the arthritic injured person, thereby reducing efflux and maintaining efficacy against methotrexate. The nobiletin and the methotrexate have obvious synergistic effect, and under the non-cytotoxic concentration of the nobiletin, the combined treatment of the nobiletin and the methotrexate can restore the growth inhibition and the apoptosis of RA-FLS/methotrexate cells. Nobiletin can be used as a potential chemosensitizer to overcome drug resistance in RA.)

1. A new use of nobiletin is characterized in that: is applied to preparing the medicine for reducing the expression of P-gp in the arthritic synovial tissue.

2. A novel use of nobiletin according to claim 1, wherein: is applied to preparing the medicine for reducing the drug resistance of RA-FLS cells to methotrexate.

3. The composition of nobiletin having a novel use according to claim 1 or 2, characterized in that: comprises 10-30 parts of nobiletin and 1 part of methotrexate by mass.

Technical Field

The invention belongs to the technical field of new application of medicines, and particularly relates to new application of nobiletin and a composition thereof.

Background

Rheumatoid Arthritis (RA) is a chronic autoimmune disease with multiple arthritis as a main clinical manifestation, has a high prevalence rate (0.32% -0.36%) and a high disability rate (60% -70%) in China, and has a poor prognosis, which seriously harms human life health. Currently, the etiology of RA is not clear, and it is thought that RA is associated with various factors such as immune system abnormality, inheritance, and infection. The basic pathological characteristics of RA patients include synovitis and angiogenesis, on one hand, synovial blood vessels proliferate and secrete a large amount of cytokines to cause synovial tissue thickening, cartilage and bone structural integrity is damaged, soft tissues around bone joints are invaded, and joint deformity is caused, on the other hand, angiogenesis is increased to invade various organs of human bodies, such as pulmonary heart, eyes, spleen, subcutaneous tissues and the like, and related organ dysfunction is caused along with the prolongation of the course of disease. The main symptoms are high disability rate such as arthralgia, swelling, stiffness, deformity and insufficiency, which not only affects the life quality of patients, but also increases the burden of families.

Methotrexate (MTX) is a classic disease-modifying antirheumatic drug recommended by the American College of Rheumatology (ACR) and the european union of rheumatology (EULAR) as the first choice for the treatment of rheumatoid arthritis because of its excellent therapeutic effect-to-price ratio. RA, a chronic disease, requires long-term administration. However, long-term administration of MTX results in decreased drug sensitivity and drug resistance, which leads to poor therapeutic effects. Therefore, in view of the complexity of RA disease and the disadvantages of MTX treatment, there is an urgent need for a treatment regimen that can increase the efficacy of treatment for refractory RA patients. Nobiletin (Nobiletin), a widely distributed polymethoxylated flavonoid derived from plants of the Citrus genus (Citrus) of the Rutaceae family (Rutaceae), has recently received much attention for its beneficial effects on human health. NOB can be derived from various citrus plants, indicating that a large number of NOB exist in nature and that their use in treating disease is an economically efficient method. NOB are now found to have a variety of biological activities, and their medical and economic values are still under development.

Disclosure of Invention

In order to solve the technical problems, the invention discloses a new application of nobiletin and a composition thereof based on that P-gp can be adjusted to overcome MTX drug resistance.

The invention relates to a new application of nobiletin in preparing a medicament for reducing P-gp expression in arthritic synovial tissue.

The novel application of the nobiletin is applied to preparing the medicine for reducing the drug resistance of RA-FLS cells to methotrexate.

The composition of the nobiletin with the new application comprises 10-30 parts of the nobiletin and 1 part of methotrexate by mass.

The combination of nobiletin and methotrexate reduces cartilage and bone damage and ameliorates swelling and arthritic damage. On the other hand, nobiletin can reverse P-gp expression in the arthritic injured person, thereby reducing efflux and maintaining efficacy against methotrexate. The nobiletin and the methotrexate have obvious synergistic effect, and under the non-cytotoxic concentration of the nobiletin, the combined treatment of the nobiletin and the methotrexate can restore the growth inhibition and the apoptosis of RA-FLS/methotrexate cells. Nobiletin can be used as a potential chemosensitizer to overcome drug resistance in RA.

Drawings

FIG. 1 Effect of NOB and MTX on the mouse Rheumatoid arthritis index. FIG. 2 effect of NOB on joint damage in RA mice; (A) using an H & E staining agent to stain ankle joint tissues, and amplifying a representative image by 100 times, wherein a red arrow indicates inflammatory cell infiltration, a green arrow indicates collagen fiber arrangement disorder, and a black arrow indicates an artery; (B) histological scores for each group. FIG. 3 is a graph of the effect of western blot and immunohistochemical detection of NOB on P-gp expression; (A) immunohistochemistry; (B) cumulative optical density value of p-gp; (C) western blot results. FIG. 4 is a graph of the effect of MTX (25, 50, 100, 250 and 500. mu.g/ml) on the viability of RA-FLS and RA-FLS/MTX at 24, 48 and 72 hours. Figure 5 flow cytometry measurement of apoptosis: (A) apoptotic results after 24 hours of treatment of RA-FLS/MTX cells with MTX (100. mu.g/ml), NOB (80. mu.M) alone and NOB in combination with MTX; (B) and (5) cell apoptosis quantification results. FIG. 6 is a graph of the effect of NOB on P-gp expression and activity; (A) expression of P-gp in RA-FLS and RA-FLS/MTX cells; (B) performing a Western blot, NOB (80. mu.M) MTX (100. mu.g/ml) or a combination thereof affecting the expression of P-gp in RA-FLS/MTX cells; (C) results of RT-PCR; (D-E) the effect of NOB on efflux activity of P-gp substrate was evaluated.

Detailed Description

In order to facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and examples, but the scope of the present invention is not limited to the following specific examples. Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Example 1

1. Experimental Material

Bovine type II collagen (Chondrex, Inc), incomplete freund's adjuvant (Chondrex, Inc), methotrexate was purchased from toronto research chemical, and nobiletin was purchased from shanghai source leaf responsibility ltd (purity > 99%). A first antibody: Anti-P-gp (ab170904), AKT (4691T), P-AKT (4060T), PI3K (4257T), P-PI3K (17366S), PKM2(4053T), HK2(2867T), LDHA (3582T), HIF-1 α (36169T), β -actin (3077T) were purchased from Abcam, USA, and Cell Signaling Technology, USA. The secondary antibody was purchased from sequoia cunninghamiae biotechnology limited in beijing. The ATP detection kit (H245), the GLUCOSE detection kit (A012-1) and the Lacate detection kit (H093) are purchased from Nanjing institute of bioengineering. Annexin V-FITC/PI apoptosis assayThe reagent kit (559763) was purchased from BD, usa. 10 XSDS-PAGE electrophoresis buffer (B1005-500ML), 10 XSDS electrotransfer buffer (B1006-500ML), and hypersensitive luminescent solution (P1010-100ML) were purchased from Beijing Polypley Gene technology, Inc. SDS-PAGE electrophoresis liquid (P0014B-10L), Western and IP cell lysate (P0013-100ML), protease inhibitor PMSF (100m M), BCA protein concentration determination kit (enhanced type), SDS-PAGE gel preparation kit (P0012A) and Quickblock^TMBlocking solution (TBSTx) was purchased from the bio-technical institute in cloudy days. Blue Plus IV Protein Marker (10-180kDa) was purchased from Beijing Quanjin Biotechnology, Inc.

2. Animal experiments

2.1 establishment of animal Experimental model

Bovine collagen type ii induced arthritis mouse model: dissolving bovine type II collagen by 0.05mol/L acetic acid solution, dropwise adding a proper amount of bovine type II collagen solution (the concentration is 2mg/ml) into incomplete Freund's adjuvant with equal volume, and fully emulsifying by a homogenizer, wherein the operation process is completely carried out on ice to keep low temperature and take the condition that the bovine type II collagen is not diffused in water as the success standard. After the tail root of the molded mouse was sterilized with 75% ethanol, 0.2 ml/mouse of the emulsified mixture was injected subcutaneously into the tail root, and after 7 days, the immunization was performed once at the same site at a dose of 0.1 ml/mouse. Next, on day 21 after the primary immunization, mice were given a booster intradermal injection of 100. mu.g of bovine type II collagen in incomplete Freund.

1.2 animal grouping and administration

28 male DBA/1J mice are adaptively fed for one week, 5 mice are randomly selected as a Normal group (Normal group), and 23 mice are remained to establish a CIA model, and only 20 mice are successfully molded (the arthritis index is more than or equal to 2). The rats successfully molded are divided into 4 groups by a random number method, wherein each group comprises 5 rats, namely a Model group (Model group), an NOB group, an MTX group and a combined group (NOB + MTX group). Every 5 animals were placed in individual cages and the room was automatically illuminated (12 hours light/dark cycle, light from 8:00 to 20:00), temperature (22-25 ℃) and relative humidity (45-50%) controlled, and food and water were given ad libitum. The administration was started 3 days after the second immunization and continued for 9 weeks. MTX group is used for gastric gavage three times a week and the TPE-CA group is used for gastric gavage three times a week and is used for gastric gavage three times a day and is used for gastric gavage of 1mg/kg, and TPE-CA group is used for gastric gavage three times a week and is used for gastric gavage of 20 mg/kg. NOB + MTX group MTX and NOB were gavaged per mouse at the same dose number as MTX and NOB alone.

1.3 Arthritis Index (AI) assessment

After the second immunization, the evaluation is started, once every three days, and the evaluation is carried out according to a 0-4 grade evaluation method. And scoring according to the degree of redness and swelling of the joint. The scoring criteria were as follows: (1) joints have no red swelling, 0 point; (2) mild redness or swelling of the little toe joint, score 1; (3) swelling and redness of the little toe joint and toes, score 2; (3) the entire paw under the ankle joint is totally swollen and reddened for 3 points; (4) the ankle joint was severely swollen and red, 4 points.

1.4 Micro-CT analysis

The entire paw of the mouse was fixed in 10% paraformaldehyde and one week later samples of the mouse forepaw and ankle were selected for micro CT (SkyScan 1176, SkyScan, belgium alterlar) scans. The scanning parameters of the Micro-CT are 50Kvp of voltage, 40W of mouse power and 6 superposed frames. After scanning and reconstruction, a three-dimensional image screenshot and bone parameter analysis will be performed.

2.5 synovial histopathological evaluation

The synovial tissue is fixed in 10% neutral formalin for at least 24 hr, dehydrated with 70%, 80%, 95% (I, II) and 100% (I, II) alcohol step by step, xylene I, II is transparent, and then paraffin embedded section is stained, HE stained, hematoxylin stained liquid is used to stain cell nucleus, eosin stained cell pulp, and finally pathological changes are observed by optical microscope. The dyeing process is as follows:

(1) taking paraffin sections for hydration: xylene I, II each 15min, 100% (I, II), 95%, 80%, 70% ethanol each 5min, and distilled water washing twice, each for 1 min.

(2) Adding hematoxylin for 10min, and washing with tap water for 2 min.

(3) Differentiating with 1% hydrochloric acid alcohol for 5-10 s.

(4) Bluing with tap water for 30 min.

(5) Counterstaining with 0.5% eosin alcohol solution for 2 min.

(6) 80% ethanol 5-10s, 95% (I, II) ethanol 2min each, 100% (I, II) ethanol 5min each, xylene I5 min, and xylene II 10 min.

(7) And (5) sealing the neutral gum.

2.6 Western blot

Total protein was extracted from the joints by RIPA lysis for 30min with protease inhibitors and phosphatase inhibitors. Protein concentration was measured using BCA protein concentration assay kit (Biyun day), and the extracted protein was stored at-80 ℃. 50 micrograms of protein were electrotransferred to PVDF membrane, separated with 10% SDS polyacrylamide gel, blocked with 5% skimmed milk for two hours, incubated overnight at 4 ℃ with the corresponding primary antibody, the secondary antibody was recovered the next day, washed 5 times with TBST for 5min each, secondary antibody (1:5000) was added, incubated 2 hours with shaking table at room temperature, recovered secondary antibody, washed 5 times with TBST for 5min each, and developed.

2.7 immunohistochemical analysis

Immunohistochemistry, also known as immunohistochemistry technology, is a research that uses antigen-antibody reaction to determine tissue intracellular antigens (polypeptides and proteins) by developing color-developing agents (fluorescein, enzymes, metal ions, isotopes) that label antibodies through chemical reaction, and performs localization, qualitative and relative quantitative studies on the tissue intracellular antigens. In order to detect the expression of P-gp in synovial tissue, an immunohistochemical method is adopted, and the specific steps are as follows:

1. 4% paraformaldehyde was fixed by perfusion, and the material was taken and placed in 20% sucrose solution (4 ℃) overnight. And (5) preparing a wax block. Slicing and pasting. Cleaning with 0.01MKPBS for 5min × 3;

2. adding 0.3% hydrogen peroxide methanol solution (methanol 80ml +0.01MKPBS 100ml + 30% hydrogen peroxide) for 30min to eliminate the effect of endogenous peroxidase, and washing with 0.01MPBS for 5min × 3;

3. adding 0.3% Triton X100 (30% Triton X100+0.01MKPBS 100ml) for 30min to increase cell permeability, and washing with 0.01MKPBS for 5min × 3;

4. adding primary antibody diluted by serum diluent (bovine serum albumin 1.00g +0.01MPBS 100ml + sodium azide 0.08g), and storing at 4 ℃ for 24-48 h; antibody was aspirated and washed with 0.01MKPBS for 5min × 3;

5. a0.01 MKPBS diluted secondary antibody was added and incubated at room temperature for 2 h. Washing with 0.01MKPBS for 5min × 3;

6. adding antibody such as ABC complex, incubating at room temperature for 2h, washing with 0.01MKPBS for 5min × 3; rapidly flushing with distilled water for three times;

7. adding color development liquid, performing immunohistochemical color development for no more than 30min, observing under microscope at intervals, sucking off the color development liquid when the cells are colored and the background color is light, rapidly flushing with distilled water for three times, and adding 0.01MKPBS to terminate the reaction;

8. after dehydration with gradient alcohol, the slides were mounted and photographed.

2.8 culture of drug-resistant cells

(1) Preparing a drug-containing culture solution: taking out corresponding volume of methotrexate mother liquor according to the drug-containing concentration required by the target of each drug administration period, adding the methotrexate mother liquor into a culture medium containing 10% of FBS and GMEM, and fully and uniformly mixing to obtain the drug administration culture medium with the target concentration.

(2) RA-FLS cells are induced by adopting a method of gradually increasing the concentration of methotrexate, the initial concentration is 10 mu g/ml, and in order to avoid the injury of apoptotic cells, cell debris and metabolites to living cells, the old culture medium is removed after 24h, and the fresh culture medium containing the same concentration of methotrexate is replaced. After cells tolerated and grew stably, cells were passaged 1: 2. The next culture period increased the drug concentration so that the cells were finally able to tolerate 250. mu.g/ml and maintained at the final concentration for 1 month before switching to no drug culture to continue the culture. Successful establishment of MTX acquired drug resistant cell line for 7 months

2.9 CCK-8 detection of drug-resistant cells IC50 and drug-resistant index

(1) Taking logarithmic growth RA-FLS, RA-FLS/MTX cells, digesting with a proper amount of pancreatin containing 10% EDTA, and preparing into single cell suspension. The cells were seeded in 96-well plates at 4000 cells per well (note that the cells were mixed while seeding). To prevent edge effects from occurring, 100uL of PBS solution was added per well in the surrounding wells.

(2 incubation at 37 ℃ for 24 hours in 5% CO; after cells were attached, gently aspirating the old medium. 200L of medium containing methotrexate at various concentrations were added to each well, with an initial concentration of RA-FLS cells of 10. mu.g/ml, and RA-FLS/MTX cells of 100. mu.g/ml. for 6 concentration gradients.

(3) After further incubation for 72h, the supernatant was carefully aspirated. Under the condition of keeping out of light, 110. mu.L of a prepared CCK-8 working solution (CCK-8 reagent: FBS-free GMEM medium: 1:10) was added to each well, and 5 blank control wells were additionally provided. And continuously incubating for 2 hours in a constant-temperature incubator in a dark place.

(4) The absorbance (OD) of each well at a wavelength of 450nm was measured by a microplate reader, and the inhibition rate and half-maximal inhibition concentration (IC50) were calculated for each concentration by Graphpad Prism 6.0 software, respectively. Resistance index (RF) ═ IC50 (RA-FLS/MTX cells)/IC 50(RA-FLS cells)

2.10 flow cytometry detection of apoptosis

(1) Collecting cells: get 10⁴The cells in the logarithmic growth phase are inoculated in a 6-well plate and put in an incubator overnight, and on the next day, after MTX and NOB respectively act on the cells for 24 hours independently and jointly, the cells are collected, and the supernatant and the digested cells need to be combined;

(2) and (3) cleaning cells: washing 2 times with pre-cooled PBS;

(3) grouping: each experiment is divided into a non-dyeing group, a single-dyeing Annexin V group, a single-dyeing PI group and a PI and Annexin V double-dyeing group, and the treatment group is subjected to double-dyeing from low to high;

(4) dyeing: the 4 Xbinding buffer was diluted to 1 Xbuffer with PBS, the residual PBS in the centrifuge tube was aspirated, 100. mu.L of 1 Xbinding buffer was added to each tube, the cells were blown with a pipette to resuspend them thoroughly, and the dye was added in the dark. Adding Annexin V or PI 5 muL into a single-dyeing group, adding Annexin V and PI 5 muL into a double-dyeing group, and gently mixing by using a pipette gun;

(5) and (3) computer detection: after incubation for 15 minutes in the dark at room temperature, 300. mu.L of binding buffer (1X) was added and mixed well, and the cell suspension was transferred to a 5mL flow tube in the dark for 1h on-machine detection on a flow cytometer.

2.11 statistical analysis of data

Results are expressed as mean ± Standard Deviation (SD). Comparisons between groups were performed using Graphpad Prism 6.0 one-way analysis of variance (ANOVA). Tukey's multiple comparisons were also used to compare the differences between groups. Values with p <0.05 were considered statistically significant. (. p <0.05,. p <0.01,. p <0.001)

3 results of the experiment

3.1 Effect of NOB in combination with MTX on the rheumatoid Arthritis Index (Arthritis Index) of CIA mice

Since the mice in the normal group exhibited normal hind legs and the arthritis score was 0, the arthritis index of the normal group was not shown in the figure. The arthritis score showed that clinical presentation and paw edema were significantly higher in CIA mice than in the normal group. At the same time, paw swelling was improved in the treated group. As shown in fig. 1, arthritis scores were reduced in the NOB group compared to the CIA group of mice. And had statistical differences (p < 0.05). Importantly, the combination of NOB and MTX significantly reduced the arthritis score from day 46 compared to the arthritis scores of the MTX and NOB groups. And had statistical differences (p < 0.05).

3.2 Effect of NOB on type II collagen-induced Joint Damage in RA mice

To further investigate the effect of NOB on CIA mice, H & E performed histological evaluation of the ankle joints of the mice. As shown in the figure, the tissue structure of the normal group is not obviously abnormal, the cartilage surface is smooth and has no infiltration, the boundary between the fat cell layer and the collagen fiber is clear, and the collagen fiber is arranged in order. However, in CIA mice we observed severe abnormalities in tissue structure, poorly defined adipose cell layers and collagen fibers, disorganized collagen fiber alignment, marked synovial hyperplasia, pannus formation, inflammatory cell infiltration and cartilage degeneration. Treatment with MTX improved these pathological changes and NOB administered alone slightly reduced the pathological manifestations. The histological score of the treatment of the MTX + NOB group was significantly lower than that of the CIA group (figure) and slightly better than that of the MTX group (p < 0.05).

3.3 Effect of NOB on P-gp expression

Western blot and immunohistochemistry were used to detect P-gp expression in synovial tissue. In FIG. 3C, P-gp expression was significantly higher in the CIA group than in the normal group. Compared with the CIA group, the expression of P-gp in the NOB group is obviously reduced. Similarly, the expression of P-gp in the MTX group was higher after treatment than in the normal group, and the expression of P-gp was down-regulated after the combination of MTX and NOB. These results suggest that NOB can reverse MTX resistance in CIA mice in synovial tissue. In this case, NOB treatment can reduce the expression of P-gp, which is closely related to drug resistance, and therefore, it is necessary to investigate the potential mechanism of NOB in drug resistance. 3.4 successful establishment of methotrexate resistant strains

RA-FLS cells are induced by a method of gradually increasing the concentration of methotrexate, the induction is started from an initial concentration of 10 mu g/ml, the drug concentration is doubled after the cells are tolerant, the final concentration is 250 mu g/ml, and the in vitro construction of the methotrexate acquired drug-resistant cell strain RA-FLS/MTX is successfully carried out for 7 months. Cytotoxicity experiments were conducted to compare the inhibitory effects of methotrexate on RA-FLS, RA-FLS/MTX cell proliferation.

The results of cytotoxicity experiments indicate that the survival rates of parent cells RA-FLS and drug-resistant cells RA-FLS/MTX are gradually reduced with the increase of the concentration of methotrexate, the inhibition effect of the methotrexate on the cell proliferation of RA-FLS is obviously stronger than that of RA-FLS/MTX cells, and P is less than 0.05. The IC50 value of methotrexate for parental cells was (46.99. + -. 2.47). mu.g/ml, the IC50 value for drug-resistant cells RA-FLS/MTX was (354.30. + -. 11.30). mu.g/ml, and the calculated RF was 7.54. The method shows that the sensitivity of RA-FLS/MTX cells to methotrexate is gradually reduced after long-time induction, obvious drug resistance is generated, and the methotrexate acquired drug-resistant cell strain is successfully constructed.

3.5 cytotoxic Effect of NOB on parental and drug-resistant cells

To assess whether the combination of NOB and MTX can synergistically kill RA-FLS/MTX cells, we treated RA-FLS/MTX cells with elevated concentrations of NOB and MTX, alone or in combination, for 48 hours. As shown, the addition of NOB caused MTX to significantly decrease the IC50 of RA-FLS/MTX cells. Treatment with a combination of NOB and MTX showed synergistic inhibition in cells of RA-FLS/MTX resistant strains of methotrexate (Table 1). These results indicate that the combination of NOB and MTX has an inhibitory effect on drug-resistant cells.

TABLE 1 IC50 values of methotrexate and nobiletin in RA cells

3.6 Effect of NOB on apoptosis of drug-resistant cells RA-FLS/MTX

To examine the effect of NOB on MTX-induced apoptosis in RA-FLS/MTX resistant cells, we used flow cytometry to examine this result. RA-FLS/MTX cells were treated for 24 hours with NOB and MTX alone or in combination and the apoptotic results were detected using Annexin V/PI assays. The results show that the apoptosis rate of the NOB + MTX group is obviously higher than that of the RA-FLS/MTX group and the MTX group. (p <0.05) these findings suggest that NOB can increase apoptosis in MTX-resistant RA-FLS/MTX cells while restoring sensitivity to drug-resistant cells.

3.7 Effect of NOB on P-gp function and expression in cells

Studies have shown that NOB and its derivatized drug-resistant cells are sensitive to different pathways. Considering that NOB can increase apoptosis of RA-FLS/MTX, we propose whether NOB can inhibit RA resistance by down-regulating P-gp expression. To confirm this hypothesis, western blots and quantitative RT-PCR were used to measure the level of P-gp. P-gp is more expressed in RA-FLS/MTX cells than in RA-FLS cells. Whether NOB affect P-gp expression and function is the first question we focus on. The results show that the use of NOB or MTX alone or in combination significantly affects the expression of P-gp and significantly reduces the level of P-gp mRNA in RA-FLS/MTX cells. Rh123 is a known substrate for P-gp and is used to monitor P-gp function. The results showed that Rh123 was accumulated in RA-FLS cells, but not in RA-FLS/MTX cells. Notably, NOB promoted Rh123 entry into RA-FLS/MTX cells, among other things. The results show that NOB paves the way for MTX to enter RA-FLS/MTX cells by partially inhibiting the activity and function of P-gp.

Discussion 4

On the one hand, it can be seen from the experimental results that the combination of NOB with MTX can reduce cartilage and bone damage in CIA mice and improve paw swelling and arthritic damage. On the other hand, the expression of P-gp in the CIA group was significantly higher than that in the normal group, while NOB reversed the expression of P-gp. On the other hand, P-gp expression in the MTX group was found to be similar to that in the CIA group. These results indicate that NOB may down-regulate P-gp expression in RA, thereby reducing the efflux of MTX and maintaining the efficacy of MTX. To further demonstrate this phenomenon, we used MTX-resistant RA-FLS/MTX cells as in vitro studies to further explore whether NOB can sensitize MTX to MDR in RA-FLS cells. A clear synergy between NOB and MTX was observed in RA-FLS/MTX cells. Notably, NOB + MTX co-treatment restored growth inhibition and apoptosis in RA-FLS/MTX cells at non-cytotoxic concentrations of NOB. Thus, the results suggest that NOB can overcome resistance in RA as a potential chemosensitizer.