2022 Impact Factor
2022 Impact Factor
Rheumatoid arthritis (RA) is described as a chronic inflammatory systemic disease that brings about persistent synovial membrane inflammation (Weyand and Goronzy, 2021). It is very common in 1 to 2% of the total population, but the etiology is still unclear (Mohamed et al., 2014). This sustained inflammation of the synovial membrane in arthritis is an important clinical feature as it causes cartilage damage and bone erosion of the joint, resulting in joint destruction (Hillen et al., 2017).
As mentioned above, the pathology of RA is indicated by inflammation in which it causes a variety of inflammatory cells to infiltrate both the pannus, and joint fluid, leading to the damage, deformation, and destruction of tissue (Sabokbar et al., 2017). Chemokine and inflammatory mediator production play an important role in the development and worsening of RA in patients (Cho et al., 2007; Digre et al., 2017). The imbalance in the levels of pro- and anti-inflammatory cytokines causes autoimmune and chronic inflammation which cause joint damage and arthritis to become progressively worse (Neumann et al., 2018).
As arthritis progresses in the joint, the macrophages in the synovial membrane and cartilage-pannus junction also promote inflammation which would then exacerbate RA (Kinne et al., 2000). They can secrete different cytokines and chemokines and activate major histocompatibility complex class-II (MHC Class-II) molecules (Bhattacharya et al., 2015). An example of a macrophage-secreted cytokine is tumor necrosis factor (TNF)-α which can affect the progression of arthritis. It regulates interleukin (IL)-1β expression which is important for the synovial fibroblast and chondrocyte production of prostaglandins and matrix metalloproteinases (MMP) and also stimulate macrophage-produced cytokines which are important factors that can worsen RA and cartilage damage (van den Berg et al., 1999; Drexler et al., 2008; Rose and Kooyman, 2016). Moreover, TNF-α can also contribute to osteoclast activation and differentiation which contributes to joint destruction (Rezaieyazdi et al., 2007).
Carrageenan can be isolated from red seaweed which is commonly known as the Irish moss or carrageen moss (Chondrus spp. and Gigartina spp.). It is a sulfated mucopolysaccharide that has been used to induce inflammation and along with it, inflammatory arthritis, footpad inflammation, and foot edema in animal models (Vinegar et al., 1987; Hansra et al., 2000). Not only with induction, in some studies, it has been used to exacerbate inflammatory pathological symptoms in models of RA (Hwang et al., 2008; Kim et al., 2021b). Injection of carrageenan into the rodent results in inflammation and production of different inflammatory mediators like TNF-α, cyclooxygenase (COX)-2, and prostaglandin E2 (PGE2) (Guay et al., 2004).
Phytoceramide (Pcer) is a ceramide that can be found or derived from plants and yeast. It is an intermediate metabolite that can be produced during dihydroceramide to ceramide conversion. Currently, synthetic phytoceramide is being used as a therapeutic agent for cancer treatment and other diseases. It has cytoprotective and immunostimulatory effects on different cell types. It has also been shown that a significant amount of Pcer is present in human oligodendroglioma cells, mouse tissue, brain, heart, liver, and astrocytic cells, which affects the development of the central nervous system and neurodegenerative disease progression. In recent studies, Pcer could be neuroprotective against glutamate-induced toxicity in cultured cortical neurons. Pcer also enhanced spatial memory in rats and cognitive function in mice (Lee et al., 2015) and increased the phosphorylated cyclic adenosine monophosphate (cAMP) responsive element binding protein and brain-derived neuroprotective factor in hippocampus of mice (Lee et al., 2013). These results suggest that Pcer may be useful for the treatment of neurodegenerative diseases. In addition, elevation of beta-amyloid-induced inflammatory factors and mitogen-activated protein (MAP) kinases were inhibited in hippocampus of mice by the treatment of Pcer (Jang et al., 2017).
Ceramide is associated with apoptosis while sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) were involved in anti-inflammatory pathways regulated in certain cell types (Hait and Maiti, 2017). It has also been suggested that the rheostat of ceramide and sphingosine-1-phosphate has an important role in the survival of tissue and cells in injury. S1P-ceramide rheostat regulates reactive oxygen species (ROS)-induced apoptotic proteins and signaling pathway, leading to apoptosis, survival, inflammation, and fibrosis in the kidney (Ueda, 2022). Besides these, there have been studies with results suggesting that the modulation of ceramide and acid sphingomyelinase could regulate rheumatoid synovial cells and arthritis. C2-ceramide inhibited the activation of AKT, MEK, and ERK1/2 in platelet-derived growth factor-stimulated synovial cells (Migita et al., 2000). The inhibition of acid sphingomyelinase ameliorated joint swelling, proinflammatory cytokines, and the IL-1β-induced activation of p38 MAPK signaling in rheumatoid arthritis (Beckmann et al., 2017; Zhao et al., 2021). As RA is regulated by the complex of pro-inflammatory cytokines, and chemokines in synovial fluid, and synovial tissues. These suggest that inhibition of the pro-inflammatory cytokines in an RA model would be a measure to treat arthritis (Hashizume and Mihara, 2011; Wojdasiewicz et al., 2014; Dey et al., 2016).
Previous studies involving phytoceramide on human fibroblast-like synoviocytes (FLS) or in an arthritis animal model have rarely been done to investigate its anti-arthritic efficacy. The present study was conducted to demonstrate the anti-arthritic effect of phytoceramide and investigate the mechanisms that underlie its effect.
Isolated FLS were obtained from the synovial tissues of RA patients undergoing joint replacement surgery as described (Kim et al., 2007). Cells were grown using cell culture reagents purchased from Gibco-Invitrogen (Carlsbad, CA, USA). The cell culture medium for FLS was Dulbecco’s modified eagle medium (low glucose) supplemented with 100 μg/mL streptomycin sulphate, 10% (vol/vol) fetal bovine serum, and 100 U/mL penicillin. In the experiments, the FLS passages used were passages 3-6.
Male Sprague-Dawley rats from Samtaco Co. (Osan, Korea) underwent acclimatization for 1 week prior to the experiment. They were kept in a controlled environment with a 12 h light/dark cycle at 23 ± 5°C and 55 ± 10% RH. The rats were given laboratory diet and water freely. The experimental procedures were conducted according to the Ewha Womans University Institutional Animal Care and Use Committee and NIH animal care guidelines.
Pcer (C18 type) was purchased from Doosan co. Glonet BU (Suwon, Korea) and Pcer C8 (N-octanoyl-phytosphingosine) was purchased from Matreya (State College, PA, USA), and carrageenan and IL-1β were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Four groups of rats were used in the study. These were the non-treated group (NOR; n=5), the carrageenan/kaolin (C/K)-induced control group (ART group; n=5), the ART group treated with 1 mg/kg of phytoceramide (C18 type, ART+Pcer1; n=5), the ART group treated with 10 mg/kg of phytoceramide (ART+Pcer10; n=5), and the ART group treated with 30 mg/kg of phytoceramide (ART+Pcer30; n=5).
Rats were induced with arthritis using C/K by following a protocol as described (Hwang et al., 2008; Villa et al., 2020). 3% Isoflurane in N2O/O2 mixture was used to anesthetize the rats, and an intra-articular injection of 100 μL of 5% C/K suspended in saline into the left knee joint was performed to induce arthritic inflammation. Parameters such as knee thickness, weight distribution ratio (WDR), and squeaking scores were used to evaluate the progress of arthritis in C/K-induced arthritic rats. Erythema, knee joint swelling, and algesia manifested and peaked on the first day after C/K injection. Starting from day 1, Pcer, which was dissolve in vegetable oil (1, 10, and 30 mg/kg, p.o.), was administered daily up to day 6 which was the last day of the experiment. All behavioral tests were blindly conducted (Fig. 1).
Figure 1. Schematic diagram of the carrageenan/kaolin-induced arthritis experimental schedules.
Knee thickness was measured daily for 6 days after C/K injection using a dial thickness gauge. Mean thickness values were measured daily in mm and are expressed against the thickness values.
Weight distribution ratio (WDR) was measured daily for 6 days after C/K injection with the use of an incapacitance meter (Ugo-basile Biological Research Apparatus Co., Comeria-Varese, Italy). The WDR is used to measure the percent of weight that is being applied by the rat on each hind leg as earlier described by Hwang et al. (2008). The rat’s paws were positioned on each mechanotransducer inside the test box of an incapacitance meter. The weight being applied were measured with four separate measurements being averaged and calculated. WDR% for one limb was obtained by dividing the weight applied by the indicated limb with the total weight applied by both limbs and multiplying the quotient by 100. Normal rats without arthritis would have a value of 50 which indicates that an equal weight is being applied by the rat on both hind limbs. As pain and arthritis progresses, this equilibrium is expected to shift towards the unaffected limb as the rat cannot put force on the affected knee due to pain.
Pain and knee rigidity caused by inflammation in arthritis was evaluated by measuring the number of squeaks elicited by extending and flexing of knee joints based on a modified method by Yu et al. (2002). This was measured daily for 6 days. The number of sounded squeaks within five flexion and extension cycles per limb were counted. A rating between 0 (no squeaking) and 1 (squeaking) were given as the limbs were flexed and extended. Each limb can obtain a score from 0 to 10 (maximum).
The knee joints were processed for histological staining by fixing the joints in 10% paraformaldehyde, dehydrating in increasing ethanol series, embedding in paraffin, and sectioning (6 µm, Finesse 325; Thermo Shandon Ltd., Cheshire, UK). Hematoxylin (Merck, Darmstadt, Germany) and 1% eosin (Sigma-Aldrich Co., MO, USA) were used to stain the slides and examine for morphological changes in the inflamed knee joints. Slide images were taken using a camera equipped microscope at 40× magnification (BX51; Olympus Ltd., Tokyo, Japan). Image analysis was performed by scoring of inflammation was performed blindly by three independent observers. The extent of inflammation in the knee joint was graded between 0 and 4; 0=normal (no infiltration), 1=minimal inflammation, 2=mild inflammation, 3=moderate inflammation, and 4=severe inflammation.
FLS were pretreated with different concentrations of Pcer (C8 type, 1, 10, and 30 µM), stimulated with IL-1β (10 ng/mL) for 6 h, and total cell lysates and nuclear extracts were prepared as described previously. The proteins were separated using 12% SDS-PAGE electrophoresis, transferred to a PVDF membrane, and incubated at 4°C with the primary antibodies (1:1,000) overnight and with the secondary antibodies (1:5,000) for 1 h. The proteins were detected using enhanced chemiluminescence (ECL) detection kits (Thermo Fisher Scientific Inc., Waltham, MA, USA). The relative amount of proteins was analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA).
Blood obtained from rats were allowed to clot and then centrifuged for 20 min at 3,500 rpm. After, the serum was separated and stored at –70°C until ELISA was to be performed. PGE2 (R&D Systems, Minneapolis, MN, USA), TNF-α, and IL-6 (BD Biosciences Pharmingen, San Diego, CA, USA) levels in serum were measured using ELISA kits following manufacturer provided protocol and as described earlier (Bang et al., 2009).
FLS in triplicate cultures were treated with different concentrations of Pcer (C8 type, 1, 10, and 30 µM) and stimulated with IL-1β (10 ng/mL) for 24 h. Supernatants were analyzed for TNF-α, IL-6, PGE2, and MAPK (RayBiotech Inc., GA, USA) with the use of ELISA kits as described earlier.
Statistical analysis was conducted by using one-way ANOVA using SPSS Ver. 13.0 (SPSS; Chicago, IL, USA) and further analyzed using Tukey’s post hoc test to see differences between groups. All experiments were performed with triplicate samples and repeated at least three times. Results are expressed as means ± standard error of the means. P values of <0.05 were considered statistically significant.
To evaluate arthritis progression in C/K-induced arthritis rats with Pcer treatment, WDR, knee thickness, squeaking score, and histological morphology were assessed as behavioral parameters. Knee joint thickness measurement is a simple method to evaluate joint edema in arthritis. In normal rats, the knee thickness was around 10 mm from the start and was consistent until the last day of the experiment. On the other hand, the knee thickness of the ART group increased significantly after C/K injection and was consistently high throughout the experiment. The Pcer treatment showed a tendency to decrease knee thickness starting on the 2nd day after arthritis induction up to the 6th day. Particularly, Pcer 30 mg/kg experimental group showed a significant decrease starting from day 4 (Fig. 2A). The number of vocalized squeaks were also measured to assess the degree of inflammatory pain in rats. A day after inducing arthritis, the number of squeaks peaked in rats that were induced with arthritis. The ART group had a consistently high squeaking score compared to the NOR group. Among the groups that received Pcer treatment, only the squeaking score for the ART+Pcer30 group had a significant decrease on the number of squeaks vocalized starting on day 6. This effect was not observed in the ART+Pcer1 and the ART+Pcer10 group (Fig. 2B). For the WDR, a score of 50% indicates equal weight being applied by the rats on each hind leg. A significant change in WDR was observed a day after C/K injection with the hind legs of the ART group having up to only 21% as its WDR in the arthritic leg. On the contrary, in the rats that were treated with 10 or 30 mg/kg Pcer, WDR did not decrease as much as in the ART group and this is significantly evident in the ART+Pcer30 group (Fig. 2C). To observe the morphology of the knee joints along with inflammatory signs, histological staining was employed. The small white box in each photo shows the area of cell infiltration. The NOR group showed no significant inflammation in the knee, but the ART group manifested severe signs of inflammation such as infiltrating inflammatory cells, pannus formation, synovial hyperplasia, cartilage degradation, and bone erosion. However, the groups with Pcer treatment showed reduced signs of inflammation when compared to the arthritis group (Fig. 2D).
Figure 2. Analysis of arthritic symptoms in the C/K-induced arthritis rats. Arthritic symptoms were induced by injecting C/K (100 μL of 5% solution) into the left knee. One day after C/K injection, Pcer was administered orally once daily for 6 days. Arthritic symptoms were determined by (A) left knee thickness, (B) squeaking scores, (C) weight distribution ratio, and (D) histological staining of hematoxylin and eosin (×40). The extent of inflammation in the knee joint was graded between 0 and 4. NOR: non-treated group; ART: C/K-treated control group; ART+Pcer1: C/K-induced and 1 mg/kg Pcer-treated groups; ART+Pcer10: C/K-induced and 10 mg/kg Pcer-treated group; and ART+Pcer30: C/K-induced and 30 mg/kg Pcer-treated group. C: cartilage, F: femur, M: meniscus, S: subchondral bone, T: tibia. The data are expressed as means ± SEM (n=5). ***p<0.001 vs. the NOR group; #p<0.05, ##p<0.01 and ###p<0.001 vs. the ART group.
Injecting C/K intra-articularly causes inflammatory mediators to increase in the synovium, inducing inflammation and destruction of the joint. In this study, inflammatory mediators TNF-α, IL-6, and PGE2 were analyzed (Fig. 3). The results revealed that the C/K injection group significantly increased the protein levels of TNF-α, IL-6, and PGE2 production when compared to the NOR group (Fig. 3A-3C). Oral treatment of Pcer significantly inhibited the rising serum level of inflammatory mediators TNF-α, IL-6, and PGE2 dose-dependently.
Figure 3. Effects of Pcer on the pro-inflammatory mediators on ELISA (A-C) and protein expression (D) in FLS. The FLS cells were stimulated by IL-1β and Pcer (C8) was applied 1 h after stimulation. 6 h after the Pcer treatment, the supernatant and cells were collected for ELISA or western blot. The representative western blot results from three independent experiments are shown. Quantification data are presented at the right side (D). The data are expressed as means ± SEM (n=3). ***p<0.001 vs. the NOR group and #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group.
IL-1β-stimulated FLS was used to examine the effects of Pcer in arthritis, in vitro. Stimulating FLS with IL-1β increased the levels of TNF-α, IL-6, and PGE2 in the supernatant (Fig. 4). The levels of TNF-α, IL-6, and PGE2 were found to be decreased dose-dependently in Pcer (C8 type)-treated FLS (Fig. 4A-4C). To further confirm the anti-inflammatory effect of Pcer, the protein expression levels of inducible nitric oxide synthase (iNOS), TNF-α , IL-6, and COX-2 were measured by Western blot. IL-1β stimulation increased the protein levels of each inflammatory mediator in FLS, however, Pcer pretreatment reduced the levels of iNOS and COX-2 in a dose-dependent manner (Fig. 4D). These results suggest that Pcer has anti-inflammatory effects in FLS.
Figure 4. Effects of Pcer on the serum levels of the pro-inflammatory mediators (A) TNF-α, (B) IL-6, and (C) PGE2 in C/K-induced arthritis rat model on ELISA. NOR: non-treated group; ART: C/K-treated control group; ART+Pcer1: C/K-induced and 1 mg/kg Pcer-treated groups; ART+Pcer10: C/K-induced and 10 mg/kg Pcer-treated group; and ART+Pcer30: C/K-induced and 30 mg/kg Pcer-treated group. The data are expressed as means ± SEM (n=3). ***p<0.001 vs. the NOR group; #p<0.05 and ##p<0.01 vs. the ART group.
The MAPK pathways are considered to be important in the cellular response to extracellular signals in arthritis especially in signal transduction (Thalhamer et al., 2008; Kim et al., 2021a). The involvement of the MAPKs such as p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in Pcer treatment of FLS was examined using ELISA and western blot (Fig. 5). After Pcer treatment (1-30 µM), MAPK phosphorylation was analyzed in stimulated FLS. In normal cells, significant MAPK phosphorylation was not observed in both intracellular protein and extracellular secretion, but stimulation with IL-1β significantly increased the phosphorylation levels of p38, ERK, and JNK (Fig. 5). Pcer treatment (30 µM) significantly suppressed p38, JNK, and MAPK phosphorylation in the supernatant after stimulation. Also, phosphorylation of p38, JNK, and ERK was decreased in a dose-dependent manner at the intracellular protein level.
Figure 5. Effect of Pcer on the phosphorylation of p38, ERK, and JNK MAPKs in FLS. The FLS cells were stimulated by IL-1β and Pcer (C8) was applied 1 h after stimulation. 6 h after the Pcer treatment, the supernatant and cells were collected. MAPK levels in FLS supernatants were measured using ELISA (A-C). Intracellular protein levels were measured using western blot (D). The representative western blot results from three independent experiments are shown. Quantification data are presented at the right side. The data are expressed as means ± SEM (n=3). **p<0.05, and ***p<0.001 vs. the NOR group and #p<0.05, ##p<0.01, and ###p<0.001 vs. the control group.
Rheumatoid arthritis (RA) is characterized by the persistence of inflammation in the synovial membrane (Han et al., 2015). Along with this, important signals for the development of arthritis include synovitis, synovial inflammation, and synovial hyperplasia (Scanzello and Goldring, 2012). Synovial fibroblast and synovial fluid increase in rheumatoid arthritis are involved in the development of RA and its invasiveness making the onset of synovitis a common symptom of both degenerative and inflammatory arthritis (Lee et al., 2007; Ospelt, 2017).
Synovial cells differentiate due to synovial inflammation, regardless of disease cause, which would lead to cartilage and bone erosion (Lee et al., 2007). In the synovium, the FLS and macrophage-like synovial cells would be activated and contribute to increasing inflammation, thus aggravating arthritis (Kinne et al., 2007; Tu et al., 2020). FLS of inflammatory joints secrete IL-6, IL-8, TNF, and IL-1, a few from the plethora of mediators they can produce, and macrophages would then infiltrate the synovial space (Bartok and Firestein, 2010).
Recently, studies have revealed that Pcer has various biological effects, such as neuroprotective and anti-inflammatory activity, as evidenced by its inhibitory effect in neuronal cells (Lee et al., 2015). Previous studies have focused on neuroprotective effects in the brain, but the current research has found direct evidence that Pcer has anti-inflammatory effects in arthritis-induced rats. Pcer significantly reduced inflammatory mediators induced by IL-1β in FLS and in rats with inflamed knee joints as well as improved RA symptoms in rats. Stimulation using IL-1β in RA-FLS has been used to analyze synovium proliferation in RA as this interleukin has a role in synovial membrane inflammation (Moran-Moguel et al., 2018). We found that Pcer significantly suppressed the production of IL-6, PGE2, and COX-2, important proinflammatory mediators, TNF-α, a mediator involved in vasodilation and edema formation, and iNOS. PGE2-increase suppression is important as due to its role in pain responses in inflammation (Gomez et al., 2013). The results presented are similar to other studies examining anti-inflammatory effects in arthritis animal models (Hashizume and Mihara, 2011; Wojdasiewicz et al., 2014; Dey et al., 2016; Sur et al., 2020).
As presented above, Pcer showed a significant TNF-α production inhibition in serum. On the other hand, matrix metalloproteinases (MMP)-1 and MMP-13 increase in IL-1β-stimulated RA-FLS was not affected by Pcer treatment (data not shown). This shows that Pcer inhibits joint inflammation but has no effect on MMP modulation.
MAPK, a major signaling in inflammatory cells, can be induced by inflammatory cytokines and lead to the expression of various MMP, adhesion molecules, and proinflammatory genes (Yamagishi et al., 2017). Studies have shown that the JNK pathway, as well as p38, is required for the MMP production of inflammatory mediator-stimulated or activated synovial fibroblasts (Nishitani et al., 2010). Thus, to further understand the mechanism on how Pcer affects FLS intracellular signaling, the MAPK pathway was analyzed. In this study, Pcer inhibited the phosphorylation of JNK and p38 in the supernatant of IL-1β-stimulated FLS but did not affect the phosphorylation of ERK. In addition, p38, JNK, and ERK phosphorylation were decreased at the intracellular protein levels. In our previous study, another anti-arthritic agent (Gintonin) displayed an anti-arthritic effect by decreasing the nuclear translocation of NF-kB/p65 through the ERK and JNK MAP kinase signaling pathways (Kim et al., 2021b). These suggest that the anti-inflammatory activity of Pcer is mediated through the regulation of the MAPK signaling pathways in IL-1β-stimulated FLS.
Our results suggest that the anti-inflammatory efficacy of Pcer may produce anti-arthritic effects by reducing the inflammatory response through the modulation of the MAPK pathway. However, one of the limitations of the study was that the anti-arthritic effect of Pcer was analyzed using mostly physical parameters in the animal model. Further studies are needed to study specific cellular and molecular mechanisms of Pcer in suppressing arthritic symptoms in animal models.
To conclude, Pcer can be used as basis of a potential therapeutic agent in the treatment of arthritis symptoms in arthritis patients. Future studies on anti-inflammatory and anti-arthritic disease drug development can focus on finding a novel drug that is based on Pcer with better efficacy and lesser risks for such diseases.
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