Introduction

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammatory lesions of joint tissues, which mainly affects small joints of the hands and feet, as well as other organs or tissues such as the lungs, heart, and nervous system [1]. The primary pathological changes of RA are erosion of cartilage and bone tissues, joint synovial cell infiltration, and synovial sputum formation [2]. Its pathogenesis may be closely related to the autoimmune response of genetically susceptible individuals caused by persistent infection of hemolytic Streptococcus or other bacterial metabolites and viruses [3]. Despite high incidence of RA, its etiology and pathogenesis are still poorly understood.

Several studies have shown that autoimmune response and inflammatory factors have a close relationship to the pathogenesis of RA [4, 5]. As important cytokines in inflammation, pro-inflammatory cytokines including tumor necrosis factor α (TNFα), matrix metalloproteinase (MMP)-3, and interleukin (IL)-12, IL‑6, and IL‑1β are considered to be the most significant mediators involved in the pathogenesis of RA [6]. Roy et al. have indicated that TLR4 is expressed on the surface of all immune cells except NK cells and B and T lymphocytes, and contributes greatly to the pathogen-specific recognition and activation of the natural immune system during the invasion of pathogenic microorganisms [7]. Besides, TLR4 has also been reported to mediate the expression of inflammatory factors in pancreatic tissue and is expressed on the membranes of macrophages and fibroblasts, which are the most important cells in the synovial tissue of RA patients [8]. Nuclear factor-κB (NF-κB) is a class of proteins that specifically bind to and promote transcription at the κB site of various gene promoters [9]. Regulation of NF-κB activation can effectively control the expression of inflammatory cytokines and participate in acute inflammatory reactions and chronic inflammatory diseases, including RA [10]. Therefore, the TLR4/NF-κB signaling pathway plays an essential role in RA.

Methotrexate (MTX) can reduce the rate of erythrocyte sedimentation and improve bone erosion, thus delaying the progression of RA [11]. MTX has become the basic drug for an RA combined regimen in clinical practice [12, 13]. Paclitaxel (PTX) extracted from the bark of Taxus cuspidata has anti-tumor activity against lung, gastric, and nasopharyngeal carcinomas [14]. PTX can not only block the mitotic cycle of cells and inhibit proliferation, but also affect a range of critical cellular immune and inflammatory activities [15]. Zhao et al. have pointed out that PTX can alleviate the degree of joint swelling in collagen-induced arthritis (CIA) rats [16]. PTX and MTX are both potential drugs for the treatment of arthritis. Nevertheless, the comparison of therapeutic efficacy and the mechanism of PTX alone or in combination with MTX on arthritis has not been studied.

In this study, a CIA rat model was established by induction of type II collagen. The therapeutic effects of PTX alone or in combination with MTX on RA were analyzed by observing pathological sections of ankle joint. The mechanism of MTX and PTX in treating RA was further studied by detecting TLR4 and p‑NF-κ-Bp65 protein expression and the contents of MMP‑3, IL‑12, IL‑6, IL‑1β, and TNFα.

Materials and methods

Animals

Female Sprague Dawley (SD) rats weighing 120–150 g were purchased from Shanghai Slack Laboratory Animal Co. Ltd. (Shanghai, China, license number: SCXX [Shanghai] 2017-0005). All animal experiments were conducted in accordance with international ethical guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Construction of the CIA rat model

A total of 70 SD rats were initially divided into a blank control group (n = 10) and a model group (n = 60). Type II collagen immunizing emulsion was prepared. Briefly, the type II collagen (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.1 mol/L glacial acetic acid to prepare a 2 mg/mL type II collagen solution, and then mixed with an equal volume of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) to prepare an emulsion. The concentration of type II collagen in the emulsion was 1 mg/mL. Then, three inoculation sites were chosen on the central axis of the back of rats in the model group, and 100 μL immunological emulsion was inoculated into each inoculation site. Meanwhile, 200 μL emulsion was injected into the root of the rat tail. After 7 days, the same dose of emulsion was inoculated at the same site to enhance the immunization. Fourteen days later, the CIA rat model with red limbs was successfully constructed.

Grouping and treatment

Successfully modeled CIA rats were randomly subdivided into a model group, an MTX group (1.5 mg/kg), a PTX low-dose group (1.5 mg/kg), a PTX medium-dose group (2.5 mg/kg), a PTX high-dose group (3.5 mg/kg), and an MTX + PTX group (MTX 1 mg/kg, PTX 3.5 mg/kg), with 10 rats in each group. The rats in the blank group and the CIA model group were routinely raised. Both MTX (M111239–500 mg, Aladdin Chemistry Co. Ltd., Shanghai, China) and PTX (P106868–10 mg, Aladdin Chemistry Co. Ltd., Shanghai, China) were administered intraperitoneally from day 1 (once a week for MTX, once every other day for PTX). The administration of the drug was terminated after 4 weeks of continuous administration in each of the above groups.

Enzyme-linked immunosorbent assay

Blood samples were taken from the tail vein of the rats in each group and centrifuged at 1500 r/min for 10 min at 4 °C. The concentrations of MMP‑3, IL‑12, IL‑6, IL‑1β, and TNFα in serum were determined using an ELISA kit according to the manufacturer’s instructions (Beijing Chenglin Biotechnology, Co. Ltd., Beijing, China).

Hematoxylin and eosin staining

Rats were sacrificed after being anesthetized with sodium pentobarbital (60 mg/kg) until muscular flaccidity. Complete ankle joint of the right hind limb was collected. Paraffin-embedded tissues were cut into 4‑μm sections, then deparaffinized in xylene and rehydrated with gradient ethanol. Then sections were stained with hematoxylin and eosin (Beyotime, Beijing, China). Results were observed under a light microscope.

Immunohistochemical staining

The freshly obtained tissues were fixed with 4% paraformaldehyde solution and embedded in a copper mold. Then tissues were sliced and routinely dewaxed into water for immunohistochemical analysis. Five fields with the strongest positive expression were selected from each slide, and the ratio of IL‑6- and IL‑1β-positive cells was calculated by a true color multi-functional cell image analysis management system (Image-Pro Plus software, Media Cybernetics, Silver Spring, Rockville, MD, USA).

Western blot

Synovial tissues of the symptomatic left hind limb joint were placed in lysis buffer (Beyotime, Shanghai, China) to collect supernatant. Proteins were measured by a BCA protein quantification kit (Thermo Scientific, Shanghai, China). Protein samples were separated by SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% skimmed milk for 1 h at room temperature, and incubated with primary antibodies (TLR4, 1: 200; P‑NFκBp65, 1: 200, Abcam, Cambridge, UK) at 4 °C overnight. After three washes in PBST, the membrane was incubated with the secondary antibody at room temperature for 90 min. Finally, immunoreactive bands were detected by the ECL kit (Zsbio, Ltd., Beijing, China). It was scanned by a UVP analyzer (Thermo Scientific, Shanghai, China) with β‑actin as an internal control to determine the relative expression of the target protein.

TdT-mediated dUTP nick-end labeling (TUNEL) assay

Cultured synovial tissue cells were dewaxed by xylene, followed by rehydration in graded alcohols. Then cells were incubated with proteinase K working solution at 37 °C for 20 min. After washing with PBS three times, TUNEL mixture solution (11684817910, Roche Ltd., Lewes, East Sussex, U.K.) was added in each group and the slides were incubated at 37 °C for 60 min in the dark. Notably, the blank group was only added 50 μL luciferin-labeled dUTP solution, and experimental groups were added 50 μL TdT +450 μL luciferin-labeled dUTP solution. Converter-POD 50 μL was added for incubation in dark at 37 °C for 30 min. Subsequently, the slides were washed three times, stained with hematoxylin, and sealed with neutral gum. Three samples were selected from each group, and the images were observed under a fluorescence microscope (Olympus, Tokyo, Japan) to calculate the apoptosis rate. Apoptosis rate = number of positive cells/total number of cells, and the average value was taken to represent the apoptosis rate of each sample.

Statistical analysis

All data were analyzed by SPSS 19.0 statistical software (IBM Corp., Armonk, NY, USA), and the measurement information was described by x ± stanard deviation. Statistical analysis was performed by one-way analysis of variance (ANOVA) and post-hoc test by the Dunnett method. The difference was considered statistically significant at P < 0.05.

Results

Ankle histopathological changes

To compare the therapeutic effects of PTX and MTX on RA, CIA rats were treated with PTX alone or in combination with MTX. The pathological condition of the right ankle joint of CIA rats was detected by H&E staining. Compared with the blank group, the typical CIA model group exhibited an incomplete synovial membrane infiltrated with numerous inflammatory cells, as well as tissue edema, structural disorder, increased vascular dilatation, and partial vascular congestion. In contrast, the histopathological damage of the five treatment groups was significantly improved (Fig. 1). Among them, PTX 3.5 mg/kg showed a relatively intact synovial structure, occasional inflammatory cell infiltration, no edema or congestion, and the number of blood vessels was at normal group level. These data indicated that both PTX and MTX improved histopathology of CIA, PTX 3.5 mg/kg having the most significant effect.

Fig. 1
figure 1

Paclitaxel (PTX) and methotrexate (MTX) improved histopathological changes in collagen-induced arthritis (CIA) rats. Histopathological changes in the right hind limb ankle joint of CIA rats were detected by hematoxylin and eosin (H&E) staining (magnification ×400; scale bar: 50 μm)

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PTX and MTX induced cell apoptosis

TUNEL assay was performed to determine the apoptotic effect of PTX and MTX either alone or in combination on synovial tissue cells. Cell apoptosis of each experimental group was markedly lower than that of the control group. In addition, the apoptosis rate of synovial tissue cells in the PTX group was more than that of the PTX and MTX combined group, with no statistical difference (Fig. 2).

Fig. 2
figure 2

Paclitaxel (PTX) and methotrexate (MTX) induced cell apoptosis. TdT mediated-dUTP Nick-End Labeling (TUNEL) assay was performed to determine the apoptotic effect of PTX and MTX either alone or in combination on synovial tissue cells (magnification ×200; scale bar: 100 μm). **P < 0.01, ***P < 0.001 compared with blank group

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PTX and MTX downregulated IL‑6 and IL‑1β expression

There were almost no positive cells in the blank group, while the rate of IL‑6- and IL‑1β-positive cells in the CIA model group was 65% and 59%, respectively, which were apparently higher than that in the blank group (Figs. 3 and 4). Compared with the model group, the number of IL‑6- and IL‑1β-positive cells in the MTX 1 mg/kg, PTX 1 mg/kg, PTX 1.5 mg/kg, PTX 3.5 mg/kg, and MTX + PTX groups were all decreased to varying degrees, accompanied by weakened positive signal intensity (42%, 58%, 51%, 23%, and 47% for IL‑6; 32%, 48%, 39%, 14%, 35% for IL‑1β, respectively), with the lowest in the PTX 3.5 mg/kg group. These findings indicated that PTX and MTX could reduce the expression rate of IL‑6- and IL‑1β-positive cells.

Fig. 3
figure 3

Paclitaxel (PTX) and methotrexate (MTX) reduced the rate of IL‑6-positive cells. IL‑6-positive cell rate in collagen-induced arthritis (CIA) rats was detected by immunohistochemistry (magnification ×400; scale bar: 50 μm). Immunohistochemistry quantification of IL‑6-positive cells. **P < 0.01 compared with blank group; #P < 0.05, ##P < 0.01 compared with model group

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Fig. 4
figure 4

Paclitaxel (PTX) and methotrexate (MTX) lowered the rate of IL‑1β-positive cells. The rate of IL‑1β-positive cells in collagen-induced arthritis (CIA) rats was examined by immunohistochemistry (magnification ×400; scale bar: 50 μm). Immunohistochemistry quantification of IL‑1β-positive cells. ***P < 0.001 compared with blank group; #P < 0.05, ###P < 0.001 compared with model group

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PTX and MTX inhibited the expression of TLR4 and p‑NF-κBp65 proteins

Western blot was conducted to confirm the impact of PTX and MTX on TLR4 and p‑NF-κBp65 protein expression. As shown in Fig. 5, the expression levels of TLR4 and p‑NF-κBp65 proteins in the CIA model group were significantly higher than those of the blank group (P < 0.001). Compared with the model group, TLR4 and p‑NF-κBp65 protein expression in the treatment groups was downregulated to different degrees, with the most significant effect in the PTX 3.5 mg/kg group (P < 0.001).

Fig. 5
figure 5

Paclitaxel (PTX) and methotrexate (MTX) inhibited TLR4 and phosphorylated (p)-κBp65 protein expression. a The expression levels of TLR4 and p‑NF-κBp65 proteins in the synovial tissue of left hind limb joints of collagen-induced arthritis (CIA) rats were detected by western blot. Quantifications of (b) TLR4 and (c) p‑NF-κBp65 protein expression. ***P < 0.001 compared with blank group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with model group

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TLR4 and p‑NF-κBp65 reduced the concentration of MMP-3, IL‑12, IL‑6, IL1-β, and TNFα

To further examine the influence of TLR4 and p‑NF-κBp65 on downstream inflammatory factors, the concentrations of MMP‑3, IL‑12, IL‑6, IL‑β, and TNF‑α were detected by ELISA. In line with the results, the MMP‑3, IL‑12, IL‑6, IL‑1β, and TNFα concentrations in the model group were significantly higher than those in the blank group (P < 0.001; Fig. 6). Compared with the model group, the concentrations of MMP‑3, IL‑12, IL‑6, IL‑β, and TNFα in the treatment groups were decreased to diverse degrees, with PTX 3.5 mg/kg showing the most obvious inhibitory effect.

Fig. 6
figure 6

TLR4 and phosphorylated (p)-NF-κBp65 reduced the concentrations of MMP‑3, IL‑12, IL‑6, IL1‑β, and TNFα. Detection of (a) MMP‑3, (b) IL‑12, (c) IL‑6, (d) IL‑1β, and (e) TNFα concentrations in serum of collagen-induced arthritis (CIA) rat tail vein by enzyme-linked immunosorbent assay (ELISA). Paclitaxel (PTX), methotrexate (MTX) ***P < 0.001 compared with blank group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with model group

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Discussion

In this study, we compared the therapeutic effects of PTX alone or in combination with MTX on RA and found that the combination of PTX and MTX was less potent than PTX 3.5 mg/kg alone. Moreover, PTX and MTX could lower the concentrations of MMP‑3, IL‑12, IL‑6, IL‑β, and TNF‑α by inhibiting TLR4 and p‑NF-κBp65 protein expression, thereby achieving the purpose of treating RA.

Currently, the major therapeutic drugs for RA are non-steroidal anti-inflammatory drugs, anti-rheumatic drugs, glucocorticoids, and biologics, which can alleviate the disease, but cannot prevent development of the disease and are accompanied by toxicity [17]. As an internationally recognized arthritis model with many similar immunological and pathological features to human RNA, CIA has become an ideal model to study the pathogenesis of RA and to screen therapeutic drugs [18]. Hence, a CIA rat model was established in this study to verify the therapeutic efficacy and related mechanisms of PTX and MTX on RA.

Previous studies have revealed the anti-cancer function of PTX in various tumors including breast and ovarian cancers. Yoshifumi et al. also found that intraperitoneal injection of PTX (6, 12, 24, and 48 mg/kg) suppressed cell proliferation and exhibited cytotoxicity toward PC-3 cells in a time-dependent manner. Among them, PTX 12 mg/kg did not result in severe leukopenia or liver damage, but still had similar anti-cancer efficacy to high-dose PTX. For cancer patients undergoing chemotherapy, PTX 135–175 mg/m2 is recommended [19]. With the exception of anti-tumor activity, recent research has identified the therapeutic impact of low-dose PTX on non-cancer human diseases such as renal and liver fibrosis and skin disorders [20,21,22]. Xu et al. suggested that PTX (1.0 and 1.5 mg/kg) could lower arthritis scores and alleviate CIA in a dose-dependent manner [23]. Besides, prophylaxis with PTX before CIA onset can completely prevent CIA, and PTX treatment after CIA onset can relieve clinical symptoms, including redness and fever. Tony et al. believed that low-dose MTX could improve the main symptoms, signs, and inflammatory exudation of RA patients [24]. These findings suggested that PTX and MTX play therapeutic roles in RA treatment. Interestingly, a study conducted by Zhao confirmed the superior therapeutic effect of PTX 2.5 mg/kg over MTX 1 mg/kg in the treatment of CIA rats [16]. However, the impact of PTX alone or in combination with MTX on RA remains unclear. Thus, different doses of PTX alone or in combination with MTX were adopted in CIA rats to compare their therapeutic effects on RA. As expected, our results demonstrated that both MTX and PTX improved the pathological features of RA and downregulated the expression levels of inflammatory factors IL‑6 and IL‑1β. Surprisingly, the effect after engagement of MTX and PTX combined therapy was weaker than that after PTX 3.5 mg/kg administration. This result might be related to the fact that MTX mainly acted on the S phase of the cell cycle and decreased G0/G1 to S phase transition in cells, while PTX mainly acted on the G2/M phase of the cell cycle [25, 26]; the combination of MTX and PTX inhibited the proliferation of inflammatory cells and excessively interfered with the mitosis of normal cells, resulting in drug-induced tissue damage. Taken together, the combination of PTX and MTX was less effective than intraperitoneal injection of PTX 3.5 mg/kg every other day, which was the most appropriate dose observed in this study.

The autoimmune response is closely associated with the pathogenesis of RA [4, 5]. It has been found that TLR4 is an important protein molecule involved in non-specific immunity and also a bridge connecting specific and non-specific immunity [27]. Intracellular signal transduction initiated by TLR4 can activate NF-κB, which is involved in the amplification of inflammatory responses by regulating the expression of inflammatory cytokines [28]. NF-κB activation is characterized by inhibition of IKBα phosphorylation and degradation. After activation, NF-κB enters nucleus and triggers the expression of specific genes and a variety of inflammatory cytokines [34]. Therefore, we speculated that the therapeutic function of PTX and MTX on RA by may be related to the expression of TLR4 and p‑NF-κBp65. To verify this idea, western blot was performed to evaluate TLR4 and p‑NF-κBp65 expression in synovial tissues of the left hind limb joints of CIA rats. Results showed that PTX and MTX dramatically inhibited TLR4 and p‑NF-κBp65 protein expression.

Numerous studies have shown that IL‑12, IL‑6, and IL1‑β can improve vascular permeability and promote angiogenesis of synovial tissues and the formation of chronic inflammation of the synovium [29, 30]. TNFα facilitates the secretion of inflammatory transmitters by synovial cells and stimulates the dilation of blood vessels, thereby enhancing the inflammatory response [31]. MMP‑3 is a major mediator involved in arthritis, contributing to the formation of fibroblasts and participation in the activation of glial proteases, leading to inflammation [32]. Zhou et al. found that inhibition of IL‑1β and TNFα expression could achieve the purpose of anti-articular inflammation, indicating that inflammatory factors are indispensable in the development of RA [33]. In this study, PTX and MTX downregulated the expression of the inflammatory factors MMP‑3, IL‑12, IL‑6, IL‑1β, and TNFα in serum. Luo et al. [34] put forward that TRL4 in peripheral blood of patients with pneumonia was positively correlated with downstream inflammatory cytokines, such as TNFα, IL‑1, IL‑6, and IL‑10. Similarly, our results showed that inhibition of TLR4 and p‑NF-κBp65 proteins expression reduced MMP‑3, IL‑12, IL‑6, IL1‑β, and TNFα levels, indicating that TLR4 and p‑NF-κBp65 expression was positively correlated with the expression of inflammatory factors in serum.

A main limitation of this study was the lack of parameters such as joint infiltration, edematous tissue, structure disorder, and quality of synovial membrane. Further research on these issues is required.

In summary, PTX and MTX have therapeutic effects on RA by downregulating TLR4 and p‑NF-κBp65 protein expression in synovial tissue of CIA model, thereby reducing the concentrations of inflammatory factors in serum. Moreover, PTX alone is superior to PTX in combination with MTX, and intraperitoneal injection of PTX 3.5 mg/kg every other day is the most optimal dose observed in this study.