Dose- and stage-dependent toxic effects of prenatal prednisone exposure on fetal articular cartilage development.

Prednisone is frequently used to treat rheumatoid diseases in pregnant women because of its high degree of safety. Whether prenatal prednisone exposure (PPE) negatively impacts fetal articular cartilage development is unclear. In this study, we simulated a clinical prednisone treatment regimen to examine the effects of different timings and doses of PPE on cartilage development in female and male fetal mice. Prednisone doses (0.25, 0.5, and 1 mg/kg/d) was administered to Kunming mice at different gestational stages (0-9 gestational days, GD0-9), mid-late gestation (GD10-18), or during the entire gestation (GD0-18) by oral gavage. The amount of matrix aggrecan (ACAN) and collagen type II a1(COL2a1), and expression of transforming growth factor ß1 (TGFß1) signaling pathway also demonstrated that the chondrocyte count and ACAN and COL2a1 expression reduced in fetal mice with early and mid-late PPE, with the reduction being more significant in the mice with early PPE than that in those with PPE at other stages. Prenatal exposure to different prednisone doses prevented the reduction of TGFß signaling pathway-related genes [TGFßR1, SMAD family member 3 (Smad3), SRY-box9 (SOX9)] as well as ACAN and COL2a1 mRNA expression levels in fetal mouse cartilage, with the most significant decrease after 1 mg/kg·d PPE. In conclusion, PPE can inhibit/restrain fetal cartilage development, with the greatest effect at higher clinical dose (1 mg/kg·d) and early stage of pregnancy (GD0-9), and the mechanism may be related to TGFß signaling pathway inhibition. The result of this study provide a theoretical and experimental foundation for the rational clinical use of prednisone.

Protective effect of lupeol on arthritis induced by type II collagen via the suppression of P13K/AKT signaling pathway in Sprague dawley rats.

To explore the therapeutic value of lupeol on collagen-induced arthritis (CIA) in rats, a rheumatoid arthritis model. Lupeol is well known pentacyclic triterpene found in various plant sources, which possess anti-inflammatory and antioxidant actions. The current study was assessed the anti-arthritic potential of lupeol and its molecular mechanisms as compared with indomethacin (Indo) in collagen-induced arthritis CIA rats. The rats were randomly alienated into five groups: Control, CIA alone, CIA + lupeol (10 mg/kg bw), CIA + Indomethacin (3 mg/kg bw), and lupeol (10 mg/kg bw) alone. The paw volume, biochemical, hematological parameters, inflammatory enzymes, and cytokines were measured. As well protein expression of apoptotic proteins, and histopathological of ankle joint were examined. Inflammatory markers, cytokines, histological changes, paw volume, and inflammation were intensely reduced and enhanced apoptosis by lupeol. Alterations in hematological parameters, rheumatoid factor, C-reactive protein, and ceruloplasmin in arthritis were reverted by lupeol. Protein expressions of Bcl-2, and P13K/Akt signaling were declined, whereas the Bax, caspssae-3, and caspase-9 were elevated. These results highlighted that lupeol suppresses P13K/Akt signaling and has a promising anti-arthritic potential for collagen-induced rheumatic arthritis treatment. Hence lupeol would be suggested as an alternative natural source with potent anti-inflammatory and apoptotic actions for chronic inflammatory disorders.

Expression of mBD4, mBD3 and CRAMP during type II collagen-induced arthritis/CIA and their association with inflammation and bone-remodeling markers.

The aim of this study was to analyze the expression of mBD4, mBD3 and CRAMP in joint of mice with type II collagen-induced arthritis/CIA and to explore its possible association with IL-10, IL-4, IFN-γ, IL-17, MMP3, RANK/RANKL/OPG and histological parameters. METHODS: CIA was induced in 44 DBA/1 J mice. The joints from mice were classified into the onset, peak and remission phase of CIA. Histological sections were stained with hematoxylin-eosin and safranin O. The expression of CRAMP, mBD-3, mBD-4, and MMP-3 was evaluated using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The expression of IL-10, IL-4, IFN-γ, IL-17, RANK/RANKL/OPG was analyzed by RT-PCR. RESULTS: We observed that inflammation and immunostained cells for CRAMP increased in the peak and remission phases compared to the control group. In addition, increments in relative expressions of CRAMP were detected for the remission phase and in IL-4 and IL-17 in the peak phase compared to the control and onset phase. In addition, an increase in IL-10 in a peak phase compared to the control, as well as the relative expression of IFN-γ in remission phase was higher than in the onset phase. This was accompanied by an increase in cartilage damage in the peak phase compared to the control. Cells immunostained to MMP3 increased in the peak phase compared to the onset and control group, and relative expression of MMP3 was detected in the peak phase compared to the onset, remission, and control group. We observed that the relative expression of RANK and RANKL in the peak phase was higher than in control and onset phase. Finally, the relative expression of OPG in the peak phase compared to the onset, remission, and control group was detected. Regarding CRAMP behavior in the different phases studied, it was positively correlated with IL-4 and RANK, and showed a negative correlation with IFN-γ, IL-17, IL-10, RANKL, OPG and RANKL/OPG ratio in the control group. Also was positively correlated with IFN-γ, IL-17, IL-4, IL-10, as well as with RANK, RANKL, and OPG in the onset and peak phases of the CIA. In the peak phase, CRAMP showed a positive association with MMP3, and we observed a direct correlation between CRAMP and IFN-γ and RANKL/OPG ratio in remission phase. mBD3 correlates positively with IFN-γ, IL-17, IL-10, RANKL, OPG and RANKL/OPG ratio, and showed a negative correlation with CRAMP, MMP3, and RANK in the control group. Also, it was directly associated with IFN-γ, IL-17, IL-4, IL-10 and RANKL in the onset phase while it was inversely associated with CRAMP, MMP-3, RANK, RANKL, and OPG in the peak phase. Finally, mBD3 was inversely correlated with MMP3 in the remission phase and was directly associated with CRAMP, IFN-γ and RANKL/OPG ratio in this phase. mBD4 was directly associated with CRAMP, IFN-γ, IL-17, IL-4, IL-10, RANKL / OPG in the onset phase, and with CRAMP, IFN-γ, IL-17, IL-4, IL-10, MMP3, RANK, RANKL and OPG in the peak phase. Finally, mBD4 was positively associated with mBD3, IFN-γ, IL-17, IL-10, RANK, RANKL OPG and RANKL/OPG in the CIA remission phase. CONCLUSIONS: Our results demonstrate that CRAMP plays an important role in CIA progress and suggest that its abundance is associated with local pro- and anti-inflammatory status. This makes us propose CRAMP as a possible contributor of bone reconstruction in the last stage of CIA.

Food antigens exacerbate intestinal damage and inflammation following the disruption of the mucosal barrier.

Food antigens are closely related to progression of inflammatory bowel disease; however, details of how they induce intestinal immune responses and causes intestinal inflammation is not yet clear. The present study aimed to examine the effects of oral collagen on the intestinal mucosa, and elucidate the mechanism of food antigen-induced enteritis. Here, we provide evidence that Aspirin (a mucosal-damaging agent) and type II collagen (CII; a food antigen) acted synergistically to disrupt the intestinal mucosal barrier, and increase intestinal permeability, which resulted in a large amount of CII entered into the lamina propria, where it interacted with the intestinal immune system, promoted intestinal inflammation, and shaped innate and adaptive immune reactions into Th1-dominant. The underlying mechanism of the CII-induced intestinal inflammation may associate with higher levels of Th1, TLR2 and TLR4, and lower levels of Th2 in the intestine of Aspirin + CII treated rats. The study indicate that compromised integrity of the intestinal barrier appears to be a prerequisite for CII-induced intestinal inflammation. The synergistic effect of food antigens and mucosal barrier injury is an important cause of intestinal inflammation. This new understanding the role of food antigen on intestinal inflammation will provide us with a new strategy for treatment and prevention of intestinal inflammation.

Berberine Delays Onset of Collagen-Induced Arthritis through T Cell Suppression.

There is evidence that berberine (BBR), a clinically relevant plant compound, ameliorates clinically apparent collagen-induced arthritis (CIA) in vivo. However, to date, there are no studies involving the use of BBR which explore its prophylactic potential in this model of rheumatoid arthritis (RA). The aim of this study was to determine if prophylactic BBR use during the preclinical phase of collagen-induced arthritis would delay arthritic symptom onset, and to characterize the cellular mechanism underlying such an effect. DBA/1J mice were injected with an emulsion of bovine type II collagen (CII) and complete Freund's adjuvant (day 0) and a booster injection of CII in incomplete Freund's adjuvant (day 18) to induce arthritis. Mice were then given i.p. injections of 1 mg/kg/day of BBR or PBS (vehicle with 0.01% DMSO) from days 0 to 28, were left untreated (CIA control), or were in a non-arthritic control group (n = 15 per group). Incidence of arthritis in BBR-treated mice was 50%, compared to 90% in both the CIA and PBS controls. Populations of B and T cells from the spleens and draining lymph nodes of mice were examined on day 14 (n = 5 per group) and day 28 (n = 10 per group). BBR-treated mice had significantly reduced populations of CD4+Th and CD4+CXCR5+ Tfh cells, and an increased proportion of Foxp3+ Treg at days 14 and 28, as well as reduced expression of co-stimulatory molecules CD28 and CD154 at both endpoints. The effect seen on T cell populations and co-stimulatory molecule expression in BBR-treated mice was not mirrored in CD19+ B cells. Additionally, BBR-treated mice experienced reduced anti-CII IgG2a and anti-CII total IgG serum concentrations. These results indicate a potential role for BBR as a prophylactic supplement for RA, and that its effect may be mediated specifically through T cell suppression. However, the cellular effector involved raises concern for BBR prophylactic use in the context of vaccine efficacy and other primary adaptive immune responses.

Associations of inflammatory markers with impaired left ventricular diastolic and systolic function in collagen-induced arthritis.

BACKGROUND: High-grade inflammation may play a pivotal role in the pathogenesis of left ventricular (LV) dysfunction. Evidence to support a role of systemic inflammation in mediating impaired LV function in experimental models of rheumatoid arthritis (RA) remains limited. The aim of the present study was to determine the effects of high-grade systemic inflammation on LV diastolic and systolic function in collagen-induced arthritis (CIA). METHODS: To induce CIA, bovine type-II collagen emulsified in incomplete Freund's adjuvant was injected at the base of the tail into 21 three-month old Sprague Dawley rats. Nine-weeks after the first immunisation, LV function was assessed by pulsed Doppler, tissue Doppler imaging and Speckle tracking echocardiography. Cardiac collagen content was determined by picrosirius red staining; circulating inflammatory markers were measured using ELISA. RESULTS: Compared to controls (n = 12), CIA rats had reduced myocardial relaxation as indexed by lateral e' (early diastolic mitral annular velocity) and e'/a' (early-to-late diastolic mitral annular velocity) and increased filling pressures as indexed by E/e'. No differences in ejection fraction and LV endocardial fractional shortening between the groups were recorded. LV global radial and circumferential strain and strain rate were reduced in CIA rats compared to controls. Higher concentrations of circulating inflammatory markers were associated with reduced lateral e', e'/a', radial and circumferential strain and strain rate. Greater collagen content was associated with increased concentrations of circulating inflammatory markers and E/e'. CONCLUSION: High-grade inflammation is associated with impaired LV diastolic function and greater myocardial deformation independent of haemodynamic load in CIA rats.

Role of microRNA-26a in cartilage injury and chondrocyte proliferation and apoptosis in rheumatoid arthritis rats by regulating expression of CTGF.

This study is carried out to investigate the role of microRNA-26a (miR-26a) in cartilage injury and chondrocyte proliferation and apoptosis in rats with rheumatoid arthritis (RA) by regulating expression of CTGF. A rat model of RA induced by type II collagen was established. The rats were assigned into normal, RA, RA + mimics negative control (NC), and RA + miR-26a mimics groups, and the cells were classified into blank, mimics NC, and miR-26a mimics groups. The degree of secondary joint swelling and arthritis index, expression of miR-26a, pathological changes, proliferation and apoptosis of chondrocytes, and expression of CTGF, interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor-α, Bax, and Bcl-2 were also determined through a series of experiments. The targeting relationship between miR-26a and CTGF was verified. Initially, downregulated miR-26a was found in cartilage tissues and inflammatory articular chondrocytes of RA rats. In addition, CTGF was determined as a direct target gene of miR-26a, and upregulation of miR-26a inhibited CTGF expression in cartilage tissues of RA rats. Furthermore, upregulation of miR-26a reduced swelling and inflammation of joints, inhibited cartilage damage, apoptosis of chondrocytes, inflammatory injury, promotes proliferation, and inhibited apoptosis of inflammatory articular chondrocytes, which may be correlated with the targeting inhibition of CTGF expression. Collectively, the results demonstrate that upregulating the expression of miR-26a could attenuate cartilage injury, stimulate the proliferation, and inhibit apoptosis of chondrocytes in RA rats.

Generation of a robust model for inducing autoimmune arthritis in Sprague Dawley rats.

INTRODUCTION: The prerequisite for any experimental model in animals is similarity with the human disease, uniformity in disease severity and incidence. In antigen-induced arthritis models it is generally recognized that the major limitation is inconsistency in terms of incidence and severity. As access to strains like DBA/1 mice or Lewis rats is difficult for resource restrained laboratories, this study aimed to establish a robust and reproducible animal model of rheumatoid arthritis (RA). METHODS: Multiple approaches were undertaken for inducing arthritis in Sprague Dawley (SD) and Wistar rats using complete Freund's adjuvant (CFA), collagen type II (CII) emulsion, or different combinations of CII with low dose CFA along with lipopolysaccharide (LPS). The development of arthritis was evaluated by measuring paw edema, arthritis score, body weight, splenic index, histopathology and radiography of paw tissues. RESULTS: The combination of CII with low dose CFA and one injection of LPS resulted in 100% incidence of arthritis with disease severity ranging from moderate to severe and results were corroborated by histopathology and radiography. DISCUSSION: In a head-to-head comparison between SD and Wistar rats, the disease profile was better sustained and consistent in SD rats, and the use of CII with low dose CFA and LPS induced features akin to human RA. Taken together, a reproducible model of arthritis was established which can be replicated in any laboratory with limited resources.

Lactobacillus fermentum PC1 has the Capacity to Attenuate Joint Inflammation in Collagen-Induced Arthritis in DBA/1 Mice.

Lactobacillus strains have shown efficacy in attenuating inflammation. This study evaluated the potential of Lactobacillus fermentum PC1 for the treatment of rheumatoid arthritis (RA) using a murine model of collagen-induced arthritis. On Day 1, healthy DBA/1 mice (six to eight weeks of age) were immunized, with 100 µg of Chicken Type 11 collagen emulsified in complete Freund's adjuvant (CFA) by intradermal injection, at the base of the tail. On Day 21, the mice were immunized intraperitoneally with 100 µg of Bovine Type11 collagen in phosphate buffered saline (PBS). On Day 28, the mice were immunized intraperitoneally with 50 µg of lipopolysaccharide (LPS). Viable L. fermentum PC1 (1 × 108 colony forming units) was given daily from Day two until the end of the experiment. From Day 21 onwards, the mice were monitored daily for clinical signs of arthritis. On Day 44, the experiment was terminated. Paws were obtained for histology and serum for cytokine assays. L. fermentum PC1-fed mice had significantly reduced paw inflammation as well as decreased synovial infiltration and less cartilage damage. Circulating serum cytokine profiles revealed decreased IL-12 and increased anti-inflammatory cytokines, namely IL-4 and IL-10. Thus, early administration of L. fermentum PC1 could prove to be a valuable therapeutic agent in the management of RA.

Comparative Evaluation of 68Ga-Citrate PET/CT and 18F-FDG PET/CT in the Diagnosis of Type II Collagen-Induced Arthritis in Rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic, symmetrical, and erosive synovitis. RA is one of the most common disabling diseases in the clinic. The main clinical intervention strategies are early diagnosis and early treatment. This study aims to predict the diagnostic value of 68Ga-citrate and 18F-FDG PET/CT in RA by comparing and analyzing the value of 68Ga-citrate and 18F-FDG PET/CT for diagnosing type II collagen-induced arthritis (CIA) in rats. Some CIA models were established. Normal rats were selected as the control group, and 23 days and 40 days were selected as the early and late time points of arthritis, respectively. The semiquantitative analysis of CIA rats was carried out with 68Ga-citrate PET/CT and 18F-FDG PET/CT, and the ratio of the maximum standardized uptake (SUVmax) values in the regions of interest (ROIs) of the hind foot ankle joint and thigh muscle was calculated and statistically analyzed. The distribution of CIA rats in vivo at the 68Ga-citrate 90 min time point was studied, and the ankle tissues were evaluated with hematoxylin and eosin (HE) staining. 68Ga-citrate PET/CT is obviously superior to 18F-FDG PET/CT for CIA imaging, and the statistical results show that the difference between the two examination methods is statistically significant (P < 0.001). The uptake of these two radiopharmaceuticals showed the same trend in arthritis rats with different scores. The distribution of 68Ga-citrate at 90 min is consistent with the trend shown by 68Ga-citrate PET/CT. 68Ga-citrate PET/CT can reflect the inflammatory activity of affected joints in CIA rats earlier and more sensitively than 18F-FDG PET/CT, and this imaging advantage continues until the later stage of inflammation. Therefore, 68Ga-citrate PET/CT is worthy of further promotion and application in the clinical diagnosis of RA.

Curculigoside exerts significant anti­arthritic effects in vivo and in vitro via regulation of the JAK/STAT/NF­κB signaling pathway.

The present study aimed to investigate the anti­arthritic effects of curculigoside isolated from the rhizome of Curculigo orchioides Gaertn in vivo and in vitro, as well as to determine the potential underlying mechanisms. A rat model of arthritis was induced with type II collagen. Arthritic rats were treated with curculigoside (50 mg/kg) and blood samples were collected to determine serum levels of tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, IL­6, IL­10, IL­12 and IL­17A. Furthermore, indices of the thymus and spleen were determined. The anti­proliferative effects of curculigoside were detected with Cell Counting kit­8 assays in rheumatoid arthritis­derived fibroblast­like synoviocyte MH7A cells. In addition, expression levels of Janus kinase (JAK)1, JAK3, signal transducer and activator of transcription (STAT)3, nuclear factor (NF)­κB p65 and its inhibitor (IκB) were determined by western blotting. The results revealed that curculigoside inhibited paw swelling and arthritis scores in type II collagen­induced arthritic (CIA) rats. Additionally, curculigoside decreased serum levels of TNF­α, IL­1ß, IL­6, IL­10, IL­12 and IL­17A in CIA rats. Curculigoside also significantly inhibited MH7A cell proliferation in a time and concentration­dependent manner. Furthermore, treatment downregulated the expression of JAK1, JAK3 and STAT3, and upregulated cytosolic nuclear factor (NF)­κB p65 and IκB. In conclusion, the results of the present study indicated that curculigoside exhibited significant anti­arthritic effects in vivo and in vitro, and the molecular mechanism may be associated with the JAK/STAT/NF­κB signaling pathway.

Cinnamaldehyde and eugenol attenuates collagen induced arthritis via reduction of free radicals and pro-inflammatory cytokines.

BACKGROUND: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease which leads to bone and cartilage erosion. Oxidative stress and pro-inflammatory cytokines plays crucial role in the pathophysiology of RA. Cinnamaldehyde and eugenol have a long history of medical use in various inflammatory disorders. PURPOSE: The drugs available for the treatment of RA are associated with various side effects. The present study was conducted to evaluate the anti-arthritic effects of cinnamaldehyde and eugenol in rat model of arthritis. METHODS: Type II collagen was intradermally injected to rats for the induction of arthritis. Cinnamaldehyde (10 and 20 mg/kg/day) and eugenol (10 and 20 mg/kg/day) were given orally for 15 days, starting from day 21 to 35. Dexamethasone treated rats served as positive control. Histological, radiological and scanning electron microscopic analysis were done to monitor the effect of compounds on collagen induced arthritis (CIA). Reactive oxygen species (ROS) formation, nitric oxide and antioxidant status were also determined. The markers of biomolecular oxidation (protein, lipid and DNA) and activities of enzymatic antioxidants (superoxide dismutase, glutathione peroxidase, catalase and glutathione reductase) were also evaluated in the joint homogenate and plasma of rats. For detecting inflammation, levels of TNF-α, IL-6 and IL-10 were monitored by ELISA. RESULTS: Our results showed anti-oxidant and anti-inflammatory effects of cinnamaldehyde and eugenol in arthritic rats. Scanning electron microscopy, histopathological and radiological findings also confirmed the anti-arthritic effects of cinnamaldehyde and eugenol. Both the compounds were effective in bringing significant decrease in the levels of ROS, nitric oxide, markers of biomolecular oxidation and increase in enzymatic and non-enzymatic antioxidants. The levels of TNF-α, IL-6 and IL-10 were also ameliorated by cinnamaldehyde and eugenol treatment. Between the two phytochemicals used, eugenol was found to be more effective than cinnamaldehyde in reducing the severity of arthritis. CONCLUSION: Cinnamaldehyde and eugenol were effective in ameliorating oxidative stress and inflammation in arthritic rats. These findings indicate that cinnamaldehdye and eugenol have a great potential to be used as an adjunct in the management of RA.

Anti-arthritic effect of pentosan polysulfate in rats with collagen-induced arthritis.

Pentosan polysulfate (PPS) is currently under investigation as a potential disease-modifying antiarthritic agent. In the present study the effects of PPS on arthritic profiles based on clinical score, ankle size, histological changes, and activity of inflammatory mediators using collagen-induced arthritic rat are reported. Model of arthritis was developed in Sprague Dawley rats by intradermal injection of bovine type II collagen emulsified with incomplete Freund's adjuvant. The rats were randomly divided into four groups: normal control, arthritic control, arthritic rats treated with PPS (at dose level 20 µg/g) and arthritic rats treated with meloxicam (2 µg/g). The treatment was continued daily until the day 30. Arthritic biomarkers (cartilage oligomeric matrix protein and tartrate-resistant acid phosphatase 5b) in synovial fluid, expression of inflammatory mediators (interleukin-1ß, and tumor necrosis factor-α) and osteoclast marker genes (cathepsin K, tartrate-resistant acid phosphatase) in synovial membrane were measured. Daily administration of PPS to the arthritic rats significantly decreased the severity of arthritis by effectively suppressing the symptoms of arthritis and improving the functional recovery based on clinical score and histopathological evidence. Intriguingly, identical downregulation pattern of arthritis profiles, biological markers as well as relative mRNA levels of osteoclast markers and cytokines were monitored in arthritic rats treated with PPS. In conclusion, PPS exerted protective effects against collagen-induced arthritis in rats. The results suggest that PPS acts as an anti-inflammatory and anti-arthritic agent in decreasing the arthritic effects in collagen-induced arthritic rats.

Anti­inflammatory effects of gambogic acid in murine collagen­induced arthritis through PI3K/Akt signaling pathway.

Garcinia angustifolia is a dry resin secreted by Garcinia cambogia, which has the functions of breaking blood, detoxifying, stopping bleeding and killing insects. It is used for the treatment of cancer and brain edema. Gambogic acid is the primary active ingredient. The present study aimed to investigate the anti­inflammatory and antiproliferative effects of gambogic acid on arthritis and the possible mechanisms. It was demonstrated that gambogic acid decreased arthritic scores in murine collagen­induced arthritic mice. The tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, IL­6 and IL­18 concentrations, and caspase­3 and caspase­9 were significantly inhibited by gambogic acid in arthritic mice. Gambogic acid decreased matrix metalloproteinases (MMP)­2, MMP­9, nuclear factor (NF)­κB and phosphorylated­p38 protein expression, and increased tissue inhibitors of matrix metalloproteases­1 (TIMP­1) protein expression in arthritic mice. Furthermore, the phosphoinositide 3­kinase (PI3K)/AKT serine/threonine kinase (Akt) signaling pathway was induced in arthritic mice treated with gambogic acid. The results suggested that gambogic acid induced anti­inflammatory effects in murine collagen­induced arthritis, through the PI3K/Akt signaling pathway, and offers future potential for application in arthritis patients.

Th17 and IL-17 Cause Acceleration of Inflammation and Fat Loss by Inducing α2-Glycoprotein 1 (AZGP1) in Rheumatoid Arthritis with High-Fat Diet.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that affects the joints. High-fat diet (HFD) is a risk factor for RA and is related to inflammation but responds minimally to medication. Given the association between HFD and inflammation, it is important to understand the function of inflammation-related T cells in RA with HFD. Collagen-induced arthritis (CIA), a model of RA, was induced in HFD mice by injection of collagen II, and metabolic markers and T cells were analyzed. The metabolic index and IgG assay results were higher in HFD-CIA mice than in nonfat diet-CIA mice. Numbers of inflammation-related T cells and macrophages, such as Th1 and Th17 cells and M1 macrophages, were higher in spleens of HFD-CIA mice. HFD-CIA mice had a high level of α2-glycoprotein 1 (Azgp1), a soluble protein that stimulates lipolysis. To examine the association between Azgp1 and Th17 cells, the reciprocal effects of Azgp1 and IL-17 on Th17 differentiation and lipid metabolism were measured. Interestingly, Azgp1 increased the Th17 population of splenocytes. Taken together, our data suggest that the acceleration of fat loss caused by Azgp1 in RA with metabolic syndrome is related to the increase of IL-17. Mice injected with the Azgp1-overexpression vector exhibited more severe CIA compared with the mock vector-injected mice.

Proarrhythmic risk and determinants of cardiac autonomic dysfunction in collagen-induced arthritis rats.

BACKGROUNDS: Patients with rheumatoid arthritis (RA) have increased risk of sudden cardiac death (SCD), which is two-fold higher than general population. The driving cause of SCD was considered due to lift-threatening arrhythmia where systemic inflammation acts as the pathophysiological basis linking RA to autonomicdysfunction. METHODS: To assess the sympathetic over-activity of "inflammatory reflex", we measured heart rate variability (HRV) in a rat collagen-induced arthritis (CIA) model, whose arthritis is induced in Lewis rats by intradermal injection of emulsion of type II collagen. Single-lead electrocardiogram (ECG) was recorded for 30 min every two days. Time and frequency-domain parameters, detrended fluctuation analysis (DFA), deceleration (DC) and acceleration capacity (AC) were analyzed. RESULTS: Compared with 9 control rats, many of HRV parameters of 9 CIA rats revealed significant different. At the beginning of arthritis, LF/HF was significant higher than controls (1st week: 2.41 ± 0.7 vs. 1.76 ± 0.6, p < 0.05; 2nd week: 2.24 ± 0.5 vs. 1.58 ± 0.5, p < 0.05) indicating intensive inflammatory reflex at the initial phase of inflammation but no significant difference was observed in the following recover phase. The similar trend of DFA parameters was noted. However, the DC appeared progressive lower despite of no significant increase of the LF/HF compared with controls since 4th week. CONCLUSIONS: We observed sympathetic over-activation of inflammatory reflex during early stage of arthritis in CIA rats. The ongoing decline of DC indicated advanced cardiac autonomic dysfunction regardless of remission of acute arthritis.

Involvement of P2X7 receptor signaling on regulating the differentiation of Th17 cells and type II collagen-induced arthritis in mice.

Interleukin (IL)-17 producing T helper (Th17) cells are major effector cells in the pathogenesis of rheumatoid arthritis (RA). The P2X7 receptor (P2X7R) has emerged as a potential site in the regulation of inflammation in RA but little is known of its functional role on the differentiation of Th17 cells. This study investigates the in vitro and in vivo effects of P2X7R on Th17 cell differentiation during type II collagen (CII) induced experimental arthritis model. In CII-treated dendritic cells (DCs) and DC/CD4+ T coculture system, pretreatment with pharmacological antagonists of P2X7R (Suramin and A-438079) caused strong inhibition of production of Th17-promoting cytokines (IL-1ß, TGF-ß1, IL-23p19 and IL-6). Exposure to CII induced the elevation of mRNAs encoding retinoic acid receptor-related orphan receptor α and γt, which were abolished by pretreatment with P2X7R antagonists. Furthermore, blocking P2X7R signaling abolished the CII-mediated increase in IL-17A. Blockade of P2X7R remarkably inhibited hind paw swelling and ameliorated pathological changes in ankle joint of the collagen-induced arthritis mice. Thus, we demonstrated a novel function for P2X7R signaling in regulating CII-induced differentiation of Th17 cells. P2X7R signaling facilitates the development of the sophisticated network of DC-derived cytokines that favors a Th17 phenotype.

Matrine Exerts a Strong Anti-Arthritic Effect on Type II Collagen-Induced Arthritis in Rats by Inhibiting Inflammatory Responses.

To investigate anti-arthritic effects of matrine isolated from the roots of S. flavescens on type II collagen-induced arthritis (CIA) in rats and to explore its related potential mechanisms, CIA rats were established and administered with matrine (20, 40 or 80 mg/kg/days, for 30 days). Subsequently, blood was collected to determine serum levels of TNF-α, IL-1ß, IL-6, IL-8, IL-17A, IL-10, MMP-2, MMP-3 and MMP-9, and hind paws and knee joints were collected for histopathological examination. Furthermore, indices of the thymus and spleen were determined, and synovial tissues were collected to determine the protein expressions of p-IκB, IκB, Cox-2 and iNOS. Our results indicated that matrine significantly suppressed inflammatory reactions and synovial tissue destruction. Matrine inhibited paw swelling, arthritis indices and weight loss in CIA rats. Additionally, matrine decreased the levels of TNF-α, IL-1ß, IL-6, IL-8, IL-17A, MMP-2, MMP-3 and MMP-9. Matrine also down-regulated expressions of p-IκB, Cox-2, and iNOS but up-regulated IκB in synovial tissues in CIA rats. The results suggested matrine possesses an anti-arthritic effect in CIA rats via inhibiting the release of pro-inflammatory cytokines and proteins that promote the NF-κB pathway.

The CII-specific autoimmune T-cell response develops in the presence of FTY720 but is regulated by enhanced Treg cells that inhibit the development of autoimmune arthritis.

BACKGROUND: Fingolimod (FTY720) is an immunomodulating drug that inhibits sphingosine-1-phosphate binding and blocks T-cell egress from lymph nodes. We analyzed the effect of FTY720 on the autoimmune T- and B-cell response in autoimmune arthritis and studied the mechanisms by which it alters the function of T cells. METHODS: Human leukocyte antigen (HLA)-DR1 humanized mice were immunized with type II collagen (CII) and treated with FTY720 three times per week for 3 weeks. Arthritis was evaluated and autoimmune T- and B-cell responses were measured using proliferation assays, enzyme-linked immunosorbent assays, HLA-DR tetramers, and flow cytometry. The functional capacity of regulatory T (Treg) cells from FTY720-treated mice was measured using an in vitro suppression assay, and the role of Treg cells in inhibiting arthritis in FTY720-treated mice was evaluated using mice treated with anti-CD25 to deplete Treg cells. RESULTS: Treatment with FTY720 delayed the onset of arthritis and significantly reduced disease incidence. FTY720 did not prevent the generation of a CII-specific autoimmune T-cell response in vivo. However, as the treatment continued, these T cells became unresponsive to restimulation with antigen in vitro, and this anergic state was reversed by addition of interleukin 2. Measurements of CD4(+)CD25(+)Foxp3(+) cells in the lymph nodes revealed that the ratio of Treg to helper T (Th) cells increased twofold in the FTY720-treated mice, and in vitro assays indicated that the regulatory function of these cells was enhanced. That FTY720 stimulation of Treg cells played a major role in arthritis inhibition was demonstrated by a loss of disease inhibition and restitution of the T-cell proliferative function after in vivo depletion of the Treg cells. CONCLUSIONS: While FTY720 affects the recirculation of lymphocytes, its ability to inhibit the development of autoimmune arthritis involves several mechanisms, including the enhancement of Treg cell function by increasing the Treg/Th ratio and increased regulatory function on a per-cell basis. FTY720 did not inhibit the development of the autoimmune T-cell response, but disease inhibition appeared to be mediated by Treg cell-mediated suppression of the CII-specific T cells. These data suggest that specific targeting of Treg cells with FTY720 may be a novel therapy for autoimmunity.

Mutation of cysteine 46 in IKK-beta increases inflammatory responses.

Activation of IκB kinase ß (IKK-ß) and nuclear factor (NF)-κB signaling contributes to cancer pathogenesis and inflammatory disease; therefore, the IKK-ß-NF-κB signaling pathway is a potential therapeutic target. Current drug design strategies focus on blocking NF-κB signaling by binding to specific cysteine residues on IKK-ß. However, mutations in IKK-ß have been found in patients who may eventually develop drug resistance. For these patients, a new generation of IKK-ß inhibitors are required to provide novel treatment options. We demonstrate in vitro that cysteine-46 (Cys-46) is an essential residue for IKK-ß kinase activity. We then validate the role of Cys-46 in the pathogenesis of inflammation using delayed-type hypersensitivity (DTH) and an IKK-ß C46A transgenic mouse model. We show that a novel IKK-ß inhibitor, dihydromyricetin (DMY), has anti-inflammatory effects on WT DTH mice but not IKK-ß C46A transgenic mice. These findings reveal the role of Cys-46 in the promotion of inflammatory responses, and suggest that Cys-46 is a novel drug-binding site for the inhibition of IKK-ß.