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. Author manuscript; available in PMC: 2013 Sep 21.
Published in final edited form as: Int J Immunopathol Pharmacol. 2012 Apr-Jun;25(2):455–466. doi: 10.1177/039463201202500215

CELASTRUS TREATMENT MODULATES ANTIGEN-INDUCED GENE EXPRESSION IN LYMPHOID CELLS OF ARTHRITIC RATS

H Yu 1, S H Venkatesha 1, S Nanjundaiah 1, L Tong 3, K D Moudgil 1,2
PMCID: PMC3779360  NIHMSID: NIHMS512576  PMID: 22697077

Summary

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of global prevalence and the disease process primarily targets the synovial joints. Despite improvements in the treatment of RA over the past decade, there still is a need for new therapeutic agents that are efficacious, less expensive, and free of severe adverse reactions. Celastrus has been used in China for centuries for the treatment of rheumatic diseases. Further, we reported previously that ethanol extract of Celastrus aculeatus Merr. (Celastrus) attenuates adjuvant-induced arthritis (AA) in rats. However, the mechanisms underlying the anti-arthritic activity of Celastrus have not yet been fully defined. We reasoned that microarray analysis might offer useful insights into the pathways and molecules targeted by Celastrus. We compared the gene expression profiles of the draining lymph node cells (LNC) of Celastrus-treated (Tc) versus water-treated (Tw) rats, and each group with untreated arthritic rats before starting any treatment (To). LNC were restimulated with mycobaterial heat shock protein-65 (Bhsp65). We identified 104 differentially expressed genes (DEG) (8 upregulated, 96 downregulated) when comparing Tc with T0 rats, in contrast to 28 (12 upregulated, 16 downregulated) when comparing Tw and T0 rats. Further, 20 genes (6 upregulated, 14 downregulated) were shared by both Tw and Tc groups. Thus, Celastrus treatment (Tc) significantly downregulated a large proportion of genes compared to controls (Tw). The DEG were mainly associated with the processes of immune response, cell proliferation and apoptosis, and signaling regulation and signaling transduction. These results provide novel insights into the mechanism of anti-arthritic activity Celastrus, and unravel potential therapeutic targets for arthritis.

Keywords: Celastrus, microarray, gene expression profile, adjuvant-induced arthritis

Introduction

Rheumatoid arthritis (RA) is an autoimmune disorder that affects 1% of world’s population and it mainly targets the synovial joints (1). Cell-mediated as well as humoral immune effector responses participate in the pathogenesis of RA (2). Various mediators of inflammation and tissue damage released by the immune cells, the synovial cells and osteoclasts orchestrate the pathogenic events in autoimmune arthritis (3–6). Although the treatment of RA has improved in the last decade, there still is a significant need for new therapeutic agents that are efficacious, less expensive, and free of severe adverse reactions (7–10). Celastrus, a Chinese medicinal herb, has been used as a folk remedy in China for centuries to treat rheumatic diseases. We have previously reported the attenuation of adjuvant-induced arthritis (AA) in rats by Celastrus aculeatus Merr. (Celastrus) (11, 12). However, the mechanisms underlying the anti-arthritic activity of Celastrus have not yet been fully defined.

AA can be induced in the Lewis (RT-11) rat by subcutaneous injection of heat-killed M. tuberculosis H37Ra (Mtb). Mycobacterial heat-shock protein 65 (Bhsp65) is one of the antigenic targets of the immune response in arthritic rats (13, 14). Considering the involvement of diverse inflammatory and immune pathways in the induction and progression of AA, we argued that microarray analysis, which can provide a snapshot of concurrent changes in the expression of a large panel of genes of interest (15), would offer useful insights into the Celastrus-targeted pathways and molecules. To this effect, we examined by microarray analysis the effect of Celastrus treatment on the expression of defined Bhsp65-induced genes in the draining lymph node cells (LNC) of arthritic rats. The arthritogenic leukocytes are activated in the antigen-draining lymph nodes following s.c. injection of Mtb to Lewis rats. From the lymph nodes, these leukocytes migrate into the synovial joints. For this reason, the study of LNC provides invaluable information about the immune events that might be modulated by Celastrus treatment of rats.

We describe here for the first time the overall differential transcriptional changes in LNC of Celastrus-treated versus control arthritic rats. These analyses allowed us to identify genes regulated by Celastrus that might be playing a major role in attenuating the progression of acute AA. In addition, our results revealed new potential targets that can be further explored for the treatment of arthritis.

Materials and methods

Animals

Male Lewis (LEW/SsNHsd) (RT-11) rats, 5 to 6-week old, were purchased from Harlan Sprague Dawley (Indianapolis, IN) and housed in an accredited animal facility at University of Maryland Baltimore (UMB). All animal handling and experimental work were carried out in accordance with the National Institutes of Health (NIH) guidelines for animal welfare, and the study was approved by the Institutional Animal Care and Use Committee (IACUC). Animals were acclimated to the holding room for at least 3 d before initiation of experimental work.

Preparation of Celastrus for the treatment of arthritic rats

Celastrus (ethanol extract of Celastrus aculeatus Merr.) was prepared as described previously (11, 12). Briefly, the powder obtained by mincing dried roots and stems of C. aculeatus was extracted for 2 hr with 75% ethanol. The extract was re-extracted twice following same procedure. The final extract was concentrated and dried. To study the anti-arthritic activity of Celastrus, the ethanol extract was dissolved in water and arthritic Lewis rats were fed at the dose of 3 g/kg body weight by using the regimen described below.

Induction of adjuvant arthritis (AA) and treatment with Celastrus

AA was induced in a cohort of Lewis rats by immunizing them subcutaneously (s.c.) at the base of the tail with 2 mg/rat of heat-killed M. tuberculosis H37Ra (Mtb) (Difco, Detroit, Michigan) emulsified in 200 µl mineral oil (Sigma-Aldrich, St. Louis, MO). Thereafter, at the onset of arthritis, one group of rats was sacrificed (T0, n=3), whereas other two groups were subjected to treatment daily for 7 days either with Celastrus (in water, administered by gavage) (TC, n=3) or with the vehicle (water, by gavage) (TW, n=3). After that, these rats were sacrificed and their draining lymph node cells (LNC) (superficial inguinal, para-aortic, and popliteal) harvested for testing.

Culture of LNC and preparation of RNA

LNC (5 × 106 cells/well) were cultured for 24 hr at 37°C in a six-well plate in vitro in serum-free HL-1 medium (Lonza, Walkersville, MD) with or without Bhsp65 (5µg/ml). Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) and purified with RNeasy Mini Kit (QiagenLtd, Crawley, UK) following the manufacturer’s instructions. Then, the concentration as well as the quality of RNA was analyzed by the NanoDrop ND-1000 (NanoDrop Technologies/Thermo Scientific, Wilmington, DE) and processed further on an RNA 6000 Nano LabChip kit (Agilent Technologies lnc., Palo Alto, CA) using Agilent 2100 Bioanalyzer.

Gene Chip hybridization

Illumina bead-based array, RatRef-12 Expression BeadChip (This chip contains over 22,000 probes based on the NCBI RefSeq database) was used to perform the whole genome expression profiling. Total RNA (50 ng/µl) was used to synthesize the second strand cDNA, which then was transcribed into biotin-labeled cRNA. Purified cRNA was hybridized onto the chip (three independent experiments, i.e., 3 gene chips/group, biological replicates) according to Illumina’s Direct Hybridization Assay protocol. Thereafter, the signal was scanned on iScan.

Data acquisition and analysis

Genome Studio version 1.6.0 was used to obtain raw Illumina expression data, which was further analyzed by Gene Spring GX. For the gene expression profile in vitro in response to Bhsp65, the level of gene expression after Bhsp65 restimulation of LNC was compared with the baseline (LNC in medium only). Further, the expression profile of T0 group was compared separately with and with TC/TW group. For analysis of ex vivo gene expression profile of arthritic rats, the gene expression of LNC in medium was compared between Celastrus-treated and water-treated rats. The differentially expressed genes (DEG) were determined by unpaired t test corrected with Benjamin-Hochberg, p value <0.05, and fold change >2.0 as cut off.

Quantitative real-time PCR (q-PCR)

The expression of a select set of differentially expressed genes (DEG) (up-/down-regulated) (Ccr1, Ccr5, Cxcl10, Lpl, Socs1, and Spp1) identified in microarray analysis was validated by quantitative real-time PCR (q-PCR) using the same RNA that was used for microarray analysis. Specific primers for the selected genes were designed according to the NCBI sequence database using the software Primer Expression 2.0 and synthesized by Sigma-Aldrich (St. Louis, MO). Each RNA sample was tested in three replicates by q-PCR and the results were presented as “fold change” compared to the respective baseline using the 2-ΔΔ Ct method as described in our previous study (14).

Statistical analysis

The microarray data were analyzed for determining the significance of the level of expression of DEG (mean “fold change”). For this purpose, unpaired t test corrected with Benjamin-Hochberg and p value <0.05 coupled with a fold change >2.0 was used as the cut off. The q-PCR data were presented as “fold change” and its significance was analyzed by a two-tailed Student’s t test with a p value < 0.05.

Results

Mtb contains multiple components, including Bhsp65, the disease-related antigen (16, 17). An immunological response to Bhsp65 has been implicated in the pathogenesis of arthritis in animals and humans (18–20). Therefore, we investigated the molecular effects of Celastrus/Water treatment of arthritic rats by testing the gene expression of the draining LNC that were restimulated in vitro with Bhsp65.

Comparative gene expression profiles of Bhsp65-restimulated LNC of Celastrus/Water-treated rats versus untreated rats at onset of arthritis (T0)

To assess the Celastrus-induced changes in gene expression during the subsequent progression of arthritis following onset of the disease, we used as reference the gene expression profile of arthritic Lewis rats with AA before starting any treatment (T0). Using ‘T0’ group as the reference, we compared the gene expression profile each of Bhsp65-restimulated LNC of Water-treated (control) (TW) or Celastrus-treated (experimental group) (TC) rats to the T0 group of rats. Overall, the gene expression at Tc time-point was markedly different from that at Tw, each compared to To (Figure 1).

Figure 1. Comparison of Bhsp65-induced gene expression profile of Celastrus (Tc)/Water-treated (Tw) rats versus rats at the onset of AA (T0).

Figure 1

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A, heatmap of Bhsp65-induced DEGs in LNC of Celastrus-fed (left) and Water-fed (right) rats with AA when compared to untreated rats before starting treatment (T0). The grey shades (light to dark) of bands represents the expression level of DEGs. Venn Diagrams show the numbers of DEGs individually in Water- or Celastrus-treated group and overlapping DEG in these two groups. ↑, upregulation of gene expression compared to T0 (gene expression of LNC cultured in Bhsp65) ; ↓ downregulation of gene expression compared to T0B, The main functional categories of 13 (Water-treated)/ 60 (Celastrus-treated) well-defined functional DEG. Dark grey section shows downregulated genes, whereas light grey section represents the upregulated genes in each category.

Specifically, a comparison of the Water-treated (TW) vs. T0 rats revealed 28 DEG (12 upregulated, 16 downregulated), and these DEG were mainly associated with the processes of immune response, cell proliferation and apoptosis, and signaling regulation and signal transduction. On the contrary, a comparison of the Celastrus-treated (TC) vs. T0 rats showed 104 DEG (8 upregulated, 96 downregulated). These DEG were related with two sub-groups of processes, one that also were found in the above-mentioned Water-treated (TW) vs. T0 group, and the other that was evident only in Celastrus-treated group (e.g., oxidation-reduction, metabolism, and transportation) (Table I). The most notable finding of the comparison of the gene expression profiles of the 3 groups of rats described above was the significant downregulation of a large proportion of DEG following Celastrus treatment compared to Water treatment, with each group compared to T0.

Table I.

The number of Bhps65-induced DEG in Celastrus-treated and vehicle (water)-treated rats compared to T0.

Gene function Tw
 Tc
UR DR UR DR
Immune response related 0 4 1 17
Cell proliferation and apoptosis 2 3 0 16
Oxidation-reduction 0 0 0 5
Metabolism 0 0 0 5
Signaling regulation and signal transduction 0 1 0 4
Transporters 0 0 0 4
Non-defined 8 7 5 39
Others 2 1 2 6
Sub-total 12 16 8 96
Total 28 104
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T0, untreated arthritic rats at onset of AA; Tw, Water-treatment; Tc, Celastrus-treatment; UR, upregulated; DR, downregulated. The DEGs in response to Bhsp65 restimulation of LNC were compared. LNC from arthritic rats that received 7-d treatment of Celastrus or water, and those from arthritic rats at T0 time point were cultured with Bhsp65 for 24h. Thereafter, the total RNA was tested for gene expression by gene chip technology. The DEGs were identified by paired comparison of Celastrus or water-treated group each versus T0 using GeneSpring GX. The p value (<0.05) corrected with Benjamin-Hochberg and fold change >2.0 were set as cut off as described in Methods section. The functional categories of these DEGs were based on the main biological processes involving them.

Comparative gene expression profiles of Bhsp65-restimulated LNC of Celastrus-treated (TC) and Water-treated control (TW) rats upon completion of treatment

We then compared the gene expression of Tw and Tc groups at the completion of treatment. Celastrus-treated rats exhibited a very different gene expression profile compared to the Water-treated rats (Figure 2). One hundred and twenty DEG were identified in Water-fed arthritic rats compared to only 13 in Celastrus-fed rats (Table II). Eleven DEG were shared by these two groups of rats (Figure 2). In control rats (TW), the Bhsp65-induced genes were functionally associated with multiple pathways such as immune activity and regulation, cell proliferation and apoptosis, binding activity, diverse metabolic pathways, and signal transduction and regulation of signaling events. The numbers and main functional group of genes whose expression was altered following Bhsp65 restimulation were compared between the two groups (Table II, III). Besides the main group of genes related to immune response listed in Table III, another smaller group of interesting genes listed therein are involved in lipid metabolism and regulation of signaling pathways.

Figure 2. Comparison of Bhsp65-induced gene expression profile of Water-treated (Tw) and Celastrus-treated (Tc) arthritic rats upon completion of treatment.

Figure 2

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A, heatmap of Bhsp65-induced DEGs in LNC of Water-fed (left) and Celastrus-fed (right) arthritic rats. The grey-shaded sections represent the expression level of DEGs. Venn Diagrams show-the numbers of DEGs individually in Water- or Celastrus-treated group, and overlapping DEG in these two groups. ↑, upregulation of gene expression compared to the corresponding baseline (gene expression of LNC cultured in medium) ; ↓ downregulation of gene expression compared to the baseline. B, The main functional categories of 91 (Water-treated)/ 13 (Celastrus-treated) well-defined functional DEG. Dark grey-colored section shows downregulated genes, whereas the light grey section represents the upregulated genes in each category.

Table II.

Comparison of Bhps65-induced gene expression in Celastrus-treated and vehicle (water)-treated rats.

Gene function Tc
 Tw
UR DR UR DR
Immune response related 9 0 43 7
Cell proliferation and apoptosis 0 1 11 3
Binding activity 0 0 2 1
Metabolism 0 1 9 4
Signaling regulation and signal transduction 0 2 1 2
Non-defined 0 0 23 6
Others 0 0 5 3
Sub-total 9 4 94 26
Total 13 120
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Tw, Water-treatment; Tc, Celastrus-treatment; UR, upregulated; DR, downregulated. The DEGs in response to Bhsp65 restimulation of LNC were compared. The gene expression of LNC cultured with Bhsp65 were compared with that of LNC in in medium (med) using the same cut off (p <0.05 corrected with Benjamin-Hochberg, fold change >2.0) as described in Methods section. The functional categories of these DEGs were based on the main biological processes involving these genes.

Table III.

Bhsp65-induced DEG in Celastrus -treated and Water-treated arthritic rats.

Gene symbol Gene name Fold change (compared to baseline)
Tw Tc
1Cd36 CD36 molecule −2.3080* ―
1Ly6d_predict
ed		 Lymphocyte antigen 6 complex, locus D
(Predicted)		 −3.0536* ―
1CD40 CD40 molecule +2.0742* ―
1IL1b Interleukin-1 beta +3.3659* ―
3IL1a Interleukin-1 beta +2.4333* +2.9682*
2IL2ra Interleukin-2 receptor subunit alpha ― +2.0568*
1Ifng Interferon gamma +10.731* ―
3Irf1 Interferon regulatory factor 1 +2.8335* +3.5967*
1Ifi44 Interferon-induced protein 44 +2.4229* ―
3Ifi47 Interferon-induced protein 47 +5.4270* +7.4566*
1Ifitm1_predi
cted		 Uncharacterized protein +2.6107* ―
1Igtp Interferon gamma-induced GTPase +3.2993* ―
1Gbp2 Interferon-induced guanylate-binding
protein 2		 +5.1357* ―
1Gbp5_predi
cted		 Gbp5 protein_predi cted +2.0709* ―
1RGD1309362 Similar to interferon-inducible GTPase +2.6164* ―
1 Spp1 Osteopontin −4.8991* ―
3Irgm Immunity-related GTPase family M
protein		 +4.5530* +6.1771*
1Socs1 Suppressor of cytokine signaling 1 +3.3085* ―
1Stat1 Signal transducer and activator of
transcription 1		 +3.4744* ―
1Stat2 Signal transducer and activator of
transcription 2		 +2.2938* ―
1Sit1 Signaling threshold-regulating
transmembrane adapter 1		 −2.3245* ―
1Ccl20 C-C motif chemokine 20 +2.2993* ―
3Cxcl1 C-X-C motif chemokine 1 +2.0073* +2.0567*
1Cxcl10 C-X-C motif chemokine 10 +18.4567* ―
1Cxcl11 C-X-C motif chemokine 11 +2.3538* ―
1Ccr1 Chemokine (C-C motif) receptor 1 –2.4165* ―
1Ccr5 Chemokine (C-C motif) receptor 5 +2.9801* ―
1Nampt Nicotinamide phosphoribosyltransferase +2.4573* ―
1Zbp1 Z-DNA-binding protein 1 +2.7961* ―
3Oasl2 2’–5’ oligoadenylate synthetase-like 2 +3.2675* +2.2771*
3C2 Complement 2 +8.9802* +9.9601*
1Lgals3 Galectin-3 –2. 6534* ―
1Klrd1 Natural killer cells antigen CD94 –3.3657* ―
1RT1-A1 RT1 class Ia, locus A1 +2.1842* ―
1RT1-N3 RT1 class Ib, locus N3 +2.5441* ―
1RT1-149 RT1 class I, locus 149 +2.1041* ―
1RT1-CE7 RT1 class I, locus CE7 +2.0527* ―
1Psmd1 26S proteasome non-ATPase regulatory
subunit 1		 +2.1029* ―
1Psme2 Proteasome activator complex subunit 2 +2.5863* ―
1Psma7 Proteasome subunit alpha type-7 +2.0089* ―
1Psma9 Proteasome subunit alpha type-9 +4.1579* ―
1Herc6 Herc6 protein +2.7818* ―
1Tap1 Antigen peptide transporter 1 +3.0453* ―
1Tapbp TAP-associated glycoprotein +2.0102* ―
3Ubd Ubiquitin D +13.058* +8.4528*
1Ube2c_predic
ted		 Uncharacterized protein +2.0290* ―
1Uchl3 Ubiquitin carboxyl-terminal hydrolase
isozyme L3		 +2.0304* ―
1Siah2 E3 ubiquitin-protein ligase SIAH2 +2.0505* ―
1Fln29 TRAF-type zinc finger domain-containing
protein 1		 +2.3887* ―
1Lta Leukotriene A-4 hydrolase +2.4297* ―
1Casp11 Caspase +2.1606* ―
Sirpa Sirpa protein alpha –2.1628* –2.9324*
Lpl Lipoprotein lipase –9.3590* –6.1837*
Rgs1 Regulator of G-protein signaling 1 +2.8558* ―
Rgs2 Regulator of G-protein signaling 2 –2.1116* –2.3047*
Rasgrp2_pred
icted		 RAS guanyl-releasing protein 2 ― –2.0735*
Sag S-arrestin –2.2818* ―
Phgdh Phosphoglycerate dehydrogenase +2.2825* ―
Cyp11b1 cytochrome P450, family 11, subfamily
B, polypeptide 1		 –2.0260* ―
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Tw, Water-treatment; Tc, Celastrus-treatment

—, not significant when compared to the corresponding baseline expression (gene expression of LNC cultured in med)

*

significant when compared to the corresponding baseline expression (p <0.05 corrected with Benjamin-Hochberg, fold change >2.0)

+

upregulation

−

downregulation; DEGs labeled with 1,2,3 are genes associated with immune response

1

DEGs only in water-treated group

2

DEGs only in Celastrus-treated group

3

DEGs both in Celastrus-treated and control (water-treated) group.

In both groups, the genes involved in immune responses comprised the largest cluster among the Bhsp65-induced genes, and these genes could be further classified into three groups according to their expression level (Table III). (a) The first group included the genes whose expression was significantly altered in Water-treated rats, but remained unchanged in Celastrus-treated rats. In the former, all genes were upregulated in their expression (Table III) except Ccr1 (C-C chemokine receptor type 1), Sit1 (Signaling threshold-regulating transmembrane adapter 1), Lgals3 (Galectin-3), Klrd (Natural killer cells antigen CD94), Ly6d_predicted (Lymphocyte antigen 6 complex, locus D), Cd36 (CD36 molecule) and Spp1 (Osteopontin). The upregulated genes are involved in costimulation (Cd40), antigen processing and presentation (e.g., Tap1, RT1-149, Psmd1), response to IFN stimulation (e.g., Ifi44, Gbp2), cell migration (Cxcl10, Cxcl11, Ccl20, Ccr5), innate immunity (e.g., Zbp1) and others (e.g., Il1b, Socs1, Nampt). (b) The second group included only one gene, IL2ra, which was significantly upregulated in Celastrus-treated rats, but was unaltered in Water-treated rats. (c) The third group included 8 genes that were differentially expressed in both Celastrus-treated and Water-treated rats. All of the 8 genes in this group showed similar direction of change (upregulation) but different level of expression between the two groups of rats.

The above data indicate that Celastrus regulated multiple arthritis-related molecular responses to achieve attenuation of the disease. The most marked reduction was in the expression of Bhsp65-induced genes related to immune responses and cellular proliferation. Further, when we compared the ex vivo gene expression of LNC in medium of Celastrus-treated vs. water-treated rats, no significant difference in the gene expression profiles was found (data not shown). These results show that the effect of Celastrus was predominantly focused on the antigen (Bhsp65)-induced genes in arthritic rats.

The gene expression data obtained from microarray analysis was validated by q-PCR using a set of 6 representative genes (Ccr1, Ccr5, Cxcl10, Lpl, Socs1, and Spp1). The data obtained by the two methods matched well (Figure 3, r2=0.8074, p<0.05), thus validating the specific changes revealed by microarray analysis in our study.

Figure 3. Correlation of gene expression analysis by microarray with that by qPCR.

Figure 3

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The level of expression of a set of 6 genes obtained from RatRef-12 Expression BeadChip (Illumina) hybridization (microarray analysis) was compared to that by qPCR. The coefficient of correlation (r2) was 0.8074 and the value was <0.05.

Discussion

It has been reported by various investigators that conventionally used anti-rheumatic drugs have distinct effects on the RA-related gene expression patterns (21–26). Methotrexate (MTX), a disease-modifying anti-rheumatic drug, has complex influences dominated by reversing the expression of anti-proliferative genes (22). Glucocorticoids, like prednisolone, effectively reduce the expression of genes that are related to inflammation (21, 23). However, there is limited information on changes in the expression of disease-related genes by natural products belonging to CAM that are being used for the treatment of arthritis in traditional systems of medicine. In this context, the present study was aimed at elucidating the molecular effects of the Chinese medicinal herb Celastrus on the expression profile of arthritis-related, antigen (Bhsp65)-induced genes in the draining lymph node cells (LNC) of arthritic rats. Although several cell types other than lymphoid cells (as in LNC) participate in the pathogenesis of arthritis, this study is focused on the gene expression profiling of LNC. Further, Hsp65 has been invoked as the target antigen in inflammatory arthritis in RA (27). Our results showing Celastrus-mediated suppression of Bhsp65-induced immune activity genes are consistent with the inhibition of the progression of arthritis following Celastrus treatment. These results also validated, albeit indirectly, that Bhsp65 is a key disease-related antigenic target in arthritis.

A comparison each of Celastrus-treated (TC)/Water-treated (TW) group with untreated rats at the onset of AA (T0) provided a broad insight into the mechanism of action of Celastrus. Using T0 as reference, Celastrus downregulated a large proportion of genes involved in different biological processes (Table I). These results emphasize that Celastrus treatment suppressed the expression of genes relating to a wide range of biological processes. Thus, the anti-arthritic activity of Celastrus involved multiple pathways. Besides affecting T cell proliferation, antigen processing and presentation, and effector immune responses, Celastrus modulated the host response to oxidation-reduction (as exemplified by the interesting set of genes: Cox8a, Etfa, Hbb, Hba-a2,MGC72973; all downregulated) as well as metabolism of lipid and glucose (as evident from altered expression of the genes Aldoa, Dhps, Vim, Upp1, Leprotl1; all down regulated).

A comparative gene expression of Tc vs. Tw at the time of completion of treatment is given in Table II, III. Of the various cytokine genes examined (Table III), Celastrus suppressed the expression of IFN-γ, a Th1 cytokine in response to Bhsp65 restimulation, suggesting that the IFN-induced responses were altered by Celastrus without having a significant effect on cytokines related to Th2 or Th17 response. Additionally, IL-1β is significantly decreased by Celastrus during restimulation with Bhsp65. The chemokine CXCL10 (IP-10) showed increased expression after Bhsp65 restimulation compared to baseline in control rats, but this increase was not evident in rats treated with Celastrus (Table III). CXCL10 is a10 kDa protein induced by IFN-γ. IFN synergizes with IL-1β or TNF-α in CXCL10 production. In this regard, inhibition of IL-1β by Celastrus might contribute to reduced production of CXCL10 in Celastrus-treated rats. Both CXCL10 and its receptor CXCR3 are abundantly expressed in RA synovium and may play a role in disease pathogenesis (28, 29). Treatment of rats with anti-CXCL10 antibody results in the suppression of AA (30). Taken together, the anti-arthritic activity of Celastrus might be mediated in part via downregulation of CXCL10. In addition, the expression of CCL20 (macrophage Inflammatory Protein-3) and CCR5, which are found to be highly expressed on the synovial membrane or joint tissue of RA patients, was downregulated by Celastrus.

A marked difference (13 versus 120) in the number of DEG after Bhsp65 restimulation of LNC of Celastrus-treated versus Water-treated control rats shows that Celastrus inhibited the expression of many more genes besides those associated with immune activity. Some of the genes related with arthritis are listed in Table III. A gene of interest encodes Lpl (lipoprotein lipase; LPL). An inverse relationship between LPL and inflammatory activity in RA has been reported (31). Our results are compatible with that study; the downregulation of Lpl was observed in control rats (Water-fed) but a relatively higher expression of Lpl was evident in Celastrus-treated rats. However, it is not clear whether the change in expression of Lpl is the cause or the result of inflammation. We also observed that Rgs1 and Rgs2 with similar function showed differential expression in Water-treated vs. Celastrus-treated rats (Table III). Celastrus-treatment relatively downregulated the expression of both Rgs1 and Rasgrp2_predicted compared to control (Water-treatment), and this effect might terminate the G-protein-driven signaling. G protein signaling coordinates signals from a large number of hormones, neurotransmitters, chemokines, and autocrine and paracrine factors. By altering the expression of regulators (Rgs1 and Rasgrp2_predicted) of G-protein signaling, Celastrus might attenuate arthritis. In regard to Sag, biologically, it protects cells from apoptosis and promotes growth of cells. We suggest that the downregulation of Sag expression contributes to the reduced proliferation of lymphocytes after antigenic restimulation and thereby, helps inhibit the pathogenic T cell response in arthritis.

Increased oxidative stress has been implicated in the pathogenesis of inflammatory, autoimmune and degenerative diseases (32–34). Accordingly, natural plant products that possess anti-oxidant and free radical-scavenging properties have been shown to provide beneficial health benefits in oxidative stress-associated diseases in experimental models (35, 36). In this context, Celastrus and its bioactive component Celastrol have been reported to possess anti-oxidant properties, and to offer beneficial outcome in oxidative-stress-induced injury (36–38). These findings in part provide support to our results showing that Celastrus can downregulate the above two groups of genes involved in oxidative phosphorylation and energy metabolism.

The above-mentioned results provide useful insights into the mechanism of action of Celastrus, which suppresses AA in the Lewis rat. In summary, the beneficial effects of Celastrus treatment in AA are attributable in part to the suppression of inflammation, the inhibition of proliferation of immune cells, and the regulation of pathogenic immune effector responses. Thus global gene expression analysis provided novel insights into the etiopathologic mechanisms underlying, AA and such expression profiling is expected to lead to better diagnosis, patient classification, and individualized therapy of RA.

Acknowledgements

We thank Jing Yin and Li Tang for their help with microarray hybridization and signal readout. This work was supported by the grant R01AT004321 from the National Institutes of Health, Bethesda, MD, USA.

Footnotes

No conflict of interest

REFERENCES

  • 1.Lipsky PE. Rheumatoid arthritis. In: A. Fauci EB, Kasper D, Hauser S, longo D, Jameson J, Loscalzo J, editors. Harrison’s Principles of Intenrnal Medicine. New York: McGraw Hill New York, NY, USA; 2008. pp. 2083–2092. [Google Scholar]
  • 2.Gorman CL, Cope AP. Immune-mediated pathways in chronic inflammatory arthritis. Best Pract Res Clin Rheumatol. 2008;22:221–238. doi: 10.1016/j.berh.2008.01.003. [DOI] [PubMed] [Google Scholar]
  • 3.Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 2010;233:233–255. doi: 10.1111/j.0105-2896.2009.00859.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Firestein GS, Zvaifler NJ. How important are T cells in chronic rheumatoid synovitis?: II. T cell-independent mechanisms from beginning to end. Arthritis Rheum. 2002;46:298–308. doi: 10.1002/art.502. [DOI] [PubMed] [Google Scholar]
  • 5.Kotake S, Yago T, Kawamoto M, Nanke Y. Role of osteoclasts and interleukin-17 in the pathogenesis of rheumatoid arthritis: crucial ‘human osteoclastology’. Journal of bone and mineral metabolism. 2012;30:125–135. doi: 10.1007/s00774-011-0321-5. [DOI] [PubMed] [Google Scholar]
  • 6.Montecucco F, Mach F. Common inflammatory mediators orchestrate pathophysiological processes in rheumatoid arthritis and atherosclerosis. Rheumatology (Oxford) 2009;48:11–22. doi: 10.1093/rheumatology/ken395. [DOI] [PubMed] [Google Scholar]
  • 7.Benucci M, Saviola G, Manfredi M, Sarzi-Puttini P, Atzeni F. Cost effectiveness analysis of disease-modifying antirheumatic drugs in rheumatoid arthritis. A systematic review literature. International journal of rheumatology. 2011;2011:845496. doi: 10.1155/2011/845496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chandrashekara S. Complementary and alternative medicine in rheumatoid arthritis. Chin J Integr Med. 2011;17:731–734. doi: 10.1007/s11655-011-0870-4. [DOI] [PubMed] [Google Scholar]
  • 9.Emery P, Dorner T. Optimising treatment in rheumatoid arthritis: a review of potential biological markers of response. Ann Rheum Dis. 2011;70:2063–2070. doi: 10.1136/ard.2010.148015. [DOI] [PubMed] [Google Scholar]
  • 10.Schabert VF, Bruce B, Ferrufino CF, Globe DR, Harrison DJ, Lingala B, et al. Disability outcomes and dose escalation with etanercept, adalimumab, and infliximab in rheumatoid arthritis patients: a US-based retrospective comparative effectiveness study. Current medical research and opinion. 2012;28:569–580. doi: 10.1185/03007995.2012.656844. [DOI] [PubMed] [Google Scholar]
  • 11.Tong L, Moudgil KD. Celastrus aculeatus Merr. suppresses the induction and progression of autoimmune arthritis by modulating immune response to heat-shock protein 65. Arthritis Res Ther. 2007;9:R70. doi: 10.1186/ar2268. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Venkatesha SH, Yu H, Rajaiah R, Tong L, Moudgil KD. Celastrus-derived celastrol suppresses autoimmune arthritis by modulating antigen-induced cellular and humoral effector responses. J Biol Chem. 2011;286:15138–15146. doi: 10.1074/jbc.M111.226365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Moudgil KD, Chang TT, Eradat H, Chen AM, Gupta RS, Brahn E, et al. Diversification of T cell responses to carboxy-terminal determinants within the 65-kD heat-shock protein is involved in regulation of autoimmune arthritis. J Exp Med. 1997;185:1307–1316. doi: 10.1084/jem.185.7.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Yu H, Yang YH, Rajaiah R, Moudgil KD. Nicotine-induced differential modulation of autoimmune arthritis in the Lewis rat involves changes in interleukin-17 and anti-cyclic citrullinated peptide antibodies. Arthritis Rheum. 2011;63:981–991. doi: 10.1002/art.30219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Gorreta F, Carbone W, Barzaghi D. Genomic profiling: cDNA arrays and oligoarrays. Methods in molecular biology (Clifton, NJ. 2012;823:89–105. doi: 10.1007/978-1-60327-216-2_7. [DOI] [PubMed] [Google Scholar]
  • 16.Quintana FJ, Carmi P, Mor F, Cohen IR. DNA fragments of the human 60-kDa heat shock protein (HSP60) vaccinate against adjuvant arthritis: identification of a regulatory HSP60 peptide. J Immunol. 2003;171:3533–3541. doi: 10.4049/jimmunol.171.7.3533. [DOI] [PubMed] [Google Scholar]
  • 17.Ulmansky R, Cohen CJ, Szafer F, Moallem E, Fridlender ZG, Kashi Y, et al. Resistance to adjuvant arthritis is due to protective antibodies against heat shock protein surface epitopes and the induction of IL-10 secretion. J Immunol. 2002;168:6463–6469. doi: 10.4049/jimmunol.168.12.6463. [DOI] [PubMed] [Google Scholar]
  • 18.Gaston JS, Life PF, Jenner PJ, Colston MJ, Bacon PA. Recognition of a mycobacteria-specific epitope in the 65-kD heat-shock protein by synovial fluid-derived T cell clones. J Exp Med. 1990;171:831–841. doi: 10.1084/jem.171.3.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Huang MN, Yu H, Moudgil KD. The involvement of heat-shock proteins in the pathogenesis of autoimmune arthritis: a critical appraisal. Semin Arthritis Rheum. 2010;40:164–175. doi: 10.1016/j.semarthrit.2009.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Mia MY, Durai M, Kim HR, Moudgil KD. Heat shock protein 65-reactive T cells are involved in the pathogenesis of non-antigenic dimethyl dioctadecyl ammonium bromide-induced arthritis. J Immunol. 2005;175:219–227. doi: 10.4049/jimmunol.175.1.219. [DOI] [PubMed] [Google Scholar]
  • 21.Cutolo M, Villaggio B, Pizzorni C, Paolino S, Moretti S, Gallo F, et al. Inflammatory gene profile in early rheumatoid arthritis and modulation by leflunomide and prednisone treatment. Ann N Y Acad Sci. 2010;1193:15–21. doi: 10.1111/j.1749-6632.2009.05297.x. [DOI] [PubMed] [Google Scholar]
  • 22.Haupl T, Yahyawi M, Lubke C, Ringe J, Rohrlach T, Burmester GR, et al. Gene expression profiling of rheumatoid arthritis synovial cells treated with antirheumatic drugs. J Biomol Screen. 2007;12:328–340. doi: 10.1177/1087057107299261. [DOI] [PubMed] [Google Scholar]
  • 23.Laan RF, Jansen TL, van Riel PL. Glucocorticosteroids in the management of rheumatoid arthritis. Rheumatology (Oxford) 1999;38:6–12. doi: 10.1093/rheumatology/38.1.6. [DOI] [PubMed] [Google Scholar]
  • 24.Lindberg J, Wijbrandts CA, van Baarsen LG, Nader G, Klareskog L, Catrina A, et al. The gene expression profile in the synovium as a predictor of the clinical response to infliximab treatment in rheumatoid arthritis. PLoS One. 2010;5:e11310. doi: 10.1371/journal.pone.0011310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Meugnier E, Coury F, Tebib J, Ferraro-Peyret C, Rome S, Bienvenu J, et al. Gene expression profiling in peripheral blood cells of patients with rheumatoid arthritis in response to anti-TNF-alpha treatments. Physiol Genomics. 2011;43:365–371. doi: 10.1152/physiolgenomics.00127.2010. [DOI] [PubMed] [Google Scholar]
  • 26.van Baarsen LG, Wijbrandts CA, Gerlag DM, Rustenburg F, van der Pouw Kraan TC, Dijkmans BA, et al. Pharmacogenomics of infliximab treatment using peripheral blood cells of patients with rheumatoid arthritis. Genes Immun. 2010;11:622–629. doi: 10.1038/gene.2010.34. [DOI] [PubMed] [Google Scholar]
  • 27.Durai M, Huang MN, Moudgil KD. Self heat-shock protein 65-mediated regulation of autoimmune arthritis. Journal of autoimmunity. 2009;33:208–213. doi: 10.1016/j.jaut.2009.09.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Lee EY, Lee ZH, Song YW. CXCL10 and autoimmune diseases. Autoimmun Rev. 2009;8:379–383. doi: 10.1016/j.autrev.2008.12.002. [DOI] [PubMed] [Google Scholar]
  • 29.Patel DD, Zachariah JP, Whichard LP. CXCR3 and CCR5 ligands in rheumatoid arthritis synovium. Clin Immunol. 2001;98:39–45. doi: 10.1006/clim.2000.4957. [DOI] [PubMed] [Google Scholar]
  • 30.Salomon I, Netzer N, Wildbaum G, Schif-Zuck S, Maor G, Karin N. Targeting the function of IFN-gamma-inducible protein 10 suppresses ongoing adjuvant arthritis. J Immunol. 2002;169:2685–2693. doi: 10.4049/jimmunol.169.5.2685. [DOI] [PubMed] [Google Scholar]
  • 31.Wallberg-Jonsson S, Dahlen G, Johnson O, Olivecrona G, Rantapaa-Dahlqvist S. Lipoprotein lipase in relation to inflammatory activity in rheumatoid arthritis. Journal of internal medicine. 1996;240:373–380. doi: 10.1046/j.1365-2796.1996.53873000.x. [DOI] [PubMed] [Google Scholar]
  • 32.Cuzzocrea S. Role of nitric oxide and reactive oxygen species in arthritis. Current pharmaceutical design. 2006;12:3551–3570. doi: 10.2174/138161206778343082. [DOI] [PubMed] [Google Scholar]
  • 33.Griffiths HR. Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease? Autoimmunity reviews. 2008;7:544–549. doi: 10.1016/j.autrev.2008.04.013. [DOI] [PubMed] [Google Scholar]
  • 34.Iborra M, Moret I, Rausell F, Bastida G, Aguas M, Cerrillo E, et al. Role of oxidative stress and antioxidant enzymes in Crohn’s disease. Biochemical Society transactions. 2011;39:1102–1106. doi: 10.1042/BST0391102. [DOI] [PubMed] [Google Scholar]
  • 35.Ardestani A, Yazdanparast R, Jamshidi S. Therapeutic effects of Teucrium polium extract on oxidative stress in pancreas of streptozotocin-induced diabetic rats. J Med Food. 2008;11:525–532. doi: 10.1089/jmf.2006.0230. [DOI] [PubMed] [Google Scholar]
  • 36.Godkar PB, Gordon RK, Ravindran A, Doctor BP. Celastrus paniculatus seed oil and organic extracts attenuate hydrogen peroxide- and glutamate-induced injury in embryonic rat forebrain neuronal cells. Phytomedicine. 2006;13:29–36. doi: 10.1016/j.phymed.2003.11.011. [DOI] [PubMed] [Google Scholar]
  • 37.Sassa H, Kogure K, Takaishi Y, Terada H. Structural basis of potent antiperoxidation activity of the triterpene celastrol in mitochondria: effect of negative membrane surface charge on lipid peroxidation. Free Radic Biol Med. 1994;17:201–207. doi: 10.1016/0891-5849(94)90075-2. [DOI] [PubMed] [Google Scholar]
  • 38.Sassa H, Takaishi Y, Terada H. The triterpene celastrol as a very potent inhibitor of lipid peroxidation in mitochondria. Biochem Biophys Res Commun. 1990;172:890–897. doi: 10.1016/0006-291x(90)90759-g. [DOI] [PubMed] [Google Scholar]

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